Multi-level degradable artificial meninx repair stent and preparation method thereof

Abstract

The invention discloses a multi-layer degradable artificial meninx repair stent and a preparation method thereof, the artificial meninx repair stent main body is of a three-layer structure, the inner layer is a directional nanofiber antibacterial layer, the middle layer is a disordered nanofiber leakage-proof layer, and the outer layer is a porous micron fiber layer with bone regeneration activity. The multi-level degradable artificial meninx repair stent designed by the invention can realize sustainable release of drugs, has the effects of promoting nerve repair and effectively preventing leakage of cerebrospinal fluid, and has blood compatibility and biocompatibility; the polypeptide has the characteristic of promoting osteogenesis, and has important application in wound healing, adhesion prevention and nerve repair tissue engineering.

Description

Technical Field

[0001] This invention relates to the fields of biomaterials and tissue engineering technology, specifically to a multi-level biodegradable artificial meningeal repair scaffold and its preparation method. Background Technology

[0002] The dura mater is a thick and tough double-layered membranous tissue located between the skull and brain tissue. The outer layer is the periosteum of the skull, loosely attached to the skullcap, and more firmly attached between the cranial sutures and the skull base. The outer layer contains abundant blood vessels, which play a role in supplying blood to the skull. The inner layer is called the meningeal layer, which is thicker and tougher than the outer layer and firmly attached to the arachnoid layer, playing a role in protecting the brain.

[0003] Dural defects are common in neurosurgical practice. They can be caused by open head injuries (industrial, transportation, war, etc.), tumor erosion, congenital meningeal defects, and other intracranial disorders. Timely repair of dural defects is crucial to prevent cerebrospinal fluid leakage, brain protrusion, and pressure compression from atmospheric forces; otherwise, they can be life-threatening. Dural defects can also lead to complications such as intracranial infections, brain adhesions, and subcutaneous effusions, frequently causing conditions like headaches and brain dysfunction. Therefore, the development and clinical application of dural repair materials are currently a key research focus.

[0004] Currently, artificial dura mater made from various materials is in clinical use, mainly falling into two categories: bio-derived materials and synthetic polymer materials. Bio-derived materials primarily include allogeneic human dura mater, xenogeneic porcine / bovine pericardium, dermal matrix, and biomembranes prepared using bovine type I collagen. Synthetic polymer materials mainly include biodegradable polyester polymers, such as polylactic acid, polyglycolic acid, polycaprolactone, and polyurethane.

[0005] Currently, multilayered artificial dura mater offers the most ideal repair results for meningeal injuries. This type of artificial dura mater simulates the physiological functions of the meninges through its layered structure. It typically consists of two layers: an inner layer made of hydrophobic material and an outer layer made of hydrophilic material. However, because the outer meningeal scaffold is prepared using an electrospinning process, the resulting artificial dura mater has a disordered fiber distribution. Fibroblasts grow slowly on this type of artificial dura mater, leading to a prolonged recovery period for the damaged meninges.

[0006] Therefore, current artificial dura mater has shortcomings in clinical applications. There is a lack of an artificial dura mater repair scaffold that is similar to the natural dura mater, has good flexibility and biocompatibility, and can effectively promote the recovery of damaged brain tissue with broad industrial prospects. Summary of the Invention

[0007] The purpose of this invention is to overcome the shortcomings of existing technologies and provide a multi-level biodegradable artificial meningeal repair scaffold and its preparation method. The multi-level biodegradable artificial meningeal repair scaffold designed by this method can achieve sustainable drug release, promote nerve repair, effectively prevent cerebrospinal fluid leakage, and has blood compatibility and biocompatibility. It also has osteogenic properties and will have important applications in wound healing, adhesion prevention, and nerve repair tissue engineering.

[0008] To achieve the above objectives, the specific technical solution is as follows: In a first aspect, the present invention provides a multi-layered biodegradable drug-loaded artificial meningeal repair scaffold, wherein the artificial meningeal repair scaffold has a three-layer structure, the inner layer being a directional nanofiber antibacterial layer, the middle layer being a disordered nanofiber leak-proof layer, and the outer layer being a porous microfiber layer with bone regeneration activity.

[0009] Furthermore, the directional nanofiber antibacterial layer is a highly directional electrospun polycaprolactone / gentamicin sulfate nanofiber near the brain tissue side, used to simulate the unidirectional collagen fibers of the natural meninges near the brain tissue side; the disordered nanofiber leak-proof layer is an electrospun disordered polycaprolactone / gentamicin sulfate nanofiber, with fiber size consistent with the inner layer, which can enhance the mechanical properties of the meningeal scaffold; the bone regeneration active porous microfiber layer is a micron-sized polycaprolactone / nanohydroxyapatite fiber that can be controlled by melt electrostatic printing near the skull side.

[0010] Furthermore, the electrospun film formed by the electrospun disordered polycaprolactone / gentamicin sulfate nanofibers has high density, prevents brain tissue adhesion, and enhances the anti-infection ability of the meningeal scaffold as an antibacterial drug carrier.

[0011] Furthermore, the micron-sized polycaprolactone / nanohydroxyapatite fiber comprises 12 layers of micron-sized fibers with an angle of 45° between adjacent layers. The porous membrane with added nanohydroxyapatite promotes the biofusion of the biodegradable meningeal scaffold with the skull.

[0012] Furthermore, the thickness of the artificial meningeal repair scaffold is 296.8 μm ± 14.1 μm. The thickness of the oriented nanofiber antibacterial layer is 42.84 μm ± 4.37 μm, and its membrane pore size ranges from 1.65 μm ± 0.26 μm. The thickness of the disordered nanofiber leak-proof layer is 42.84 μm ± 4.37 μm, and its membrane pore size ranges from 1.02 μm ± 0.07 μm. The thickness of the bone regeneration-active porous microfiber layer is 211.13 μm ± 16.59 μm, and its membrane pore size ranges from 144.50 μm ± 19.79 μm.

[0013] Secondly, the present invention provides a method for preparing a multi-layered biodegradable drug-loaded artificial dura mater repair scaffold, the specific steps of which are as follows: (1) Preparation of polycaprolactone, polycaprolactone / gentamicin sulfate electrospinning solution and polycaprolactone, polycaprolactone / nanohydroxyapatite electrospinning particles: ① Polycaprolactone spinning solution: Prepare a polycaprolactone spinning solution with a content of 15 w / v% using HFIP as solvent; ② Polycaprolactone / Gentamicin Sulfate Electrospinning Solution: Using HFIP as the solvent, the added gentamicin sulfate was uniformly dispersed using a magnetic stirrer and ultrasonic cleaner. Then, polycaprolactone particles were added. The final concentrations of polycaprolactone and gentamicin sulfate in the polycaprolactone / gentamicin sulfate electrospinning solution were 15 w / v% and 1.5 w / v%, respectively. ③ Polycaprolactone electrostatic printing particles: Bio-grade polycaprolactone particles are selected; ④ Polycaprolactone / nano-hydroxyapatite electrostatic printing particles: Using HFIP as a solvent, the nano-hydroxyapatite particles added first are completely dissolved and uniformly dispersed using a magnetic stirrer. Then, polycaprolactone particles are added, and after uniform dispersion again, the HFIP is completely evaporated. The polycaprolactone / nano-hydroxyapatite film is cut into polycaprolactone / nano-hydroxyapatite electrostatic printing particles, wherein the mass ratio of nano-hydroxyapatite particles to polycaprolactone particles is 1:10. (2) The prepared polycaprolactone / gentamicin sulfate electrospinning solution was loaded into a syringe and clamped onto a single-channel precision injection pump. A layer of tin foil was pasted on the roller receiving device for easy collection. The syringe needle tip was connected to the positive terminal, the roller receiving device was grounded, and the electrospinning parameters were set as follows: voltage 13 kV, flow rate 1.2 mL / h, receiving distance 11 mm, rotation speed 3600 r / min, and electrospinning time 30 min. (3) Adjust the electrospinning parameters to 120 r / min, set the electrospinning parameters to 13 kV, 1.2 mL / h, and 11 mm receiving distance, and continue electrospinning for 90 min; (4) Stop electrospinning, place the obtained film in a fume hood to allow the solvent to evaporate fully, then cut it into 50mm×50mm pieces and paste it onto the glass, and then fix it on the motion receiving platform of the molten electrostatic printing equipment; (5) After loading polycaprolactone / nanohydroxyapatite solid particles into a microsyringe, clamp it onto a high-precision injection pump, turn on the heating switch, set the heating temperature to 85℃, after the particles melt, turn on the injection pump control software, set the flow rate to 30μL / h, after the flow rate stabilizes, adjust the receiving distance to 5 mm, adjust the high voltage power supply to 5 kV, and after forming a Taylor cone at the needle tip, make the three-dimensional platform move according to the pre-programmed program. The angle of each fiber layer changes by 45 degrees, for a total of 12 layers. The molten electrostatically printed fibers are deposited on the electrospun film to obtain a biodegradable drug-loaded artificial meningeal repair scaffold.

[0014] Thirdly, the present invention also provides an application of a multi-layered biodegradable drug-loaded artificial dura mater repair scaffold in bio-tissue engineering. The multi-layered biodegradable drug-loaded artificial dura mater repair scaffold is manufactured by adding gentamicin sulfate and nano-hydroxyapatite to polycaprolactone fibers to create a three-layered biodegradable drug-loaded dura mater scaffold, thereby achieving sustainable drug release, promoting nerve repair, effectively preventing cerebrospinal fluid leakage, having blood compatibility and biocompatibility, and having osteogenic properties.

[0015] Compared with existing technologies, the present invention has the following advantages: By simulating the microstructure and composition characteristics of the natural meninges, the present invention designs a three-layer biodegradable drug-loaded meningeal scaffold comprising a directional nanofiber antibacterial layer, a disordered nanofiber leak-proof layer, and a porous microfiber layer with bone regeneration activity. This scaffold is 3D printed in one piece, resulting in a more stable and safer structure that better conforms to the microstructure of the natural meninges, enabling sustainable drug release and promoting nerve repair; effectively preventing cerebrospinal fluid leakage; possessing blood compatibility and biocompatibility; and exhibiting osteogenic properties. Attached Figure Description

[0016] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0017] Figure 1 This is a schematic diagram of a multi-level biodegradable artificial meningeal repair scaffold structure. Figure 2 A diagram illustrating the electrostatic manufacturing process of a multi-level biodegradable artificial meningeal repair scaffold; Figure 3 Macroscopic diagram of the skull side of the PCL / GS / nHA artificial meningeal repair scaffold; Figure 4 Macroscopic diagram of the brain tissue side of the PCL / GS / nHA artificial meningeal repair scaffold; Figure 5 SEM image of the three-layer microstructure of the PCL / GS / nHA artificial meningeal repair scaffold; Figure 6 This is a diagram showing the diameter distribution of ordered fibers from electrospun fibers. Figure 7 This is a diagram showing the orientation distribution of ordered fibers in electrospinning. Figure 8 This is a diagram showing the diameter distribution of disordered fibers from electrospinning. Figure 9 This is a diagram showing the directional distribution of disordered fibers in electrospinning. Figure 10 The results of the N and S elemental analysis are shown in the figure. Figure 11 Graphs showing the XRD results of PCL and PCL / nHA; Figure 12 Graphs showing the FTIR detection results of PCL and PCL / nHA; Figure 13 Alizarin red staining results for fused electrostatically printed cranial PCL and PCL / nHA artificial meningeal repair scaffolds; Figure 14 Figure 1 shows the elemental analysis results of PCL / GS / nHA artificial meningeal repair scaffold detected by EDS. Figure 15 Elemental distribution map of PCL / GS / nHA artificial meningeal repair scaffold on the skull side, obtained by EDS analysis; Figure 16 Elemental distribution map of the brain tissue side of the PCL / GS / nHA artificial meningeal repair scaffold, obtained by EDS analysis; Figure 17 The image shows the results of drug release analysis for the artificial meningeal repair scaffold. Figure 18 Figure showing the hydrostatic pressure analysis results of the artificial meningeal repair scaffold; Figure 19 Figure 1 shows the stress-strain curve and tensile test results of the artificial meningeal repair scaffold. Figure 20 Figure 1 shows the results of tensile strength and elongation at break of the artificial meningeal repair scaffold. Figure 21 The image shows the test results of the puncture strength of the artificial meningeal repair scaffold. Figure 22 The image shows the suture strength test results of the artificial meningeal repair scaffold. Figure 23 The image shows the interlayer bonding strength test results of the PCL / GS / nHA artificial meningeal repair scaffold. Figure 24The antibacterial effect of natural seawater on artificial meningeal repair scaffolds with different drug loading for 4 days. Figure 25 The image shows the inhibition zone of natural seawater on artificial meningeal repair scaffolds with different drug loadings over 4 consecutive days. Figure 26 Electron micrographs of artificial meningeal repair scaffolds with different drug loadings after 4 days of co-culture with natural seawater. Figure 27 The image shows the hemolysis evaluation results of artificial meningeal repair scaffolds containing different concentrations of glucose. Figure 28 The CCK-8 assay results for L929 cells cultured for 1, 4, and 7 days are shown in the figure. Figure 29 Images showing cell viability and death staining results after 1d, 4d, and 7d of L929 culture in each group; Figure 30 The graph shows the cell viability results of each group after culturing L929 cells for 1 day, 4 days, and 7 days. Figure 31 ALP staining images of HuBMSCs cultured on PCL and PCL / nHA artificial meningeal repair scaffolds at 7 and 14 days. Figure 32 ALP quantification results of HuBMSCs cultured on PCL and PCL / nHA artificial meningeal repair scaffolds for 7 and 14 days; Figure 33 Images showing the skeletal staining and immunofluorescence staining results of osteogenic-related proteins COL-I and OCN in each group after 7 and 14 days of culturing HuBMSCs on PCL and PCL / nHA artificial meningeal repair scaffolds; Figure 34 Figure 1 shows the COL-I expression results of HuBMSCs cultured on PCL and PCL / nHA artificial meningeal repair scaffolds after 7 and 14 days. Figure 35 Figure 1: OCN expression results of HuBMSCs cultured on PCL and PCL / nHA artificial meningeal repair scaffolds after 7 and 14 days; Figure 36 Figure 1 shows the expression levels of osteogenic-related genes after 7 and 14 days of culturing HuBMSCs on PCL and PCL / nHA artificial meningeal repair scaffolds. Detailed Implementation

[0018] The present invention is further illustrated below by way of embodiments, but these embodiments are not intended to limit the invention to their scope. Experimental methods not specifically described in the following embodiments were performed according to conventional methods and conditions, or as selected in the product manual. All software used in these embodiments is commercially available software or open-source free software.

[0019] Example 1: Fabrication of a multi-layered biodegradable artificial dura mater repair scaffold 1. Design of artificial meningeal repair scaffold By simulating the microstructure and composition characteristics of natural meninges, a three-layer biodegradable drug-loaded meningeal scaffold was designed, comprising a directional nanofiber antibacterial layer, a disordered nanofiber leak-proof layer, and a porous microfiber layer with bone regeneration activity. As shown in Figure 1, a biodegradable drug-loaded meningeal scaffold with a three-layer structure was fabricated by adding gentamicin sulfate and nano-hydroxyapatite (PCL / GS / nHA) to polycaprolactone fibers.

[0020] like Figure 1 As shown, highly oriented electrospun polycaprolactone / gentamicin sulfate (PCL / GS) nanofibers near the brain tissue are used to mimic the unidirectional collagen fibers of the natural meninges near the brain tissue. To enhance the mechanical properties of the meningeal scaffold, the middle layer consists of electrospun disordered PCL / GS nanofibers with the same fiber size as the oriented layer. The high density of the electrospun film can prevent brain tissue adhesion and provide mechanical strength, while the addition of antibacterial drugs enhances the meningeal scaffold's resistance to infection. The micron-sized polycaprolactone / nano-hydroxyapatite (PCL / nHA) scaffold, controllably fabricated by melt electroprinting near the skull, contains 12 layers of micron-sized fibers with an angle of 45° between adjacent layers. The porous film with added nano-hydroxyapatite can promote the biofusion of the biodegradable meningeal scaffold with the skull.

[0021] 2. Manufacturing of artificial meningeal repair scaffolds (1) Preparation of PCL, PCL / GS electrospinning solution and PCL, PCL / nHA electrospinning particles.

[0022] ① PCL spinning solution: Prepare a PCL spinning solution with a content of 15 w / v% using HFIP as the solvent.

[0023] ② PCL / GS electrospinning solution: Using HFIP as solvent, the added GS was uniformly dispersed using a magnetic stirrer and an ultrasonic cleaner, and then PCL particles were added. The concentrations of PCL and GS in the final PCL / GS electrospinning solution were 15 w / v% and 1.5 w / v, respectively.

[0024] ③ PCL electrostatic printing particles: Bio-grade PCL particles purchased from Daigang Biotechnology were selected.

[0025] ④ PCL / nHA electrostatic printing particles: Using HFIP as a solvent, the added nHA particles are completely dissolved and uniformly dispersed using a magnetic stirrer. Then, PCL particles are added, and after uniform dispersion again, the HFIP is completely evaporated. The PCL / nHA film is then cut into PCL / nHA electrostatic printing particles, wherein the mass ratio of nHA particles to PCL particles is 1:10.

[0026] (2) The manufacturing process of highly oriented electrospinning PCL / GS or PCL nanofiber layers near the brain tissue side is shown in part a of Figure 2. The specific parameters are shown in Table 1.

[0027] Table 1. Process parameters for oriented and disordered PCL, PCL / GS electrospinning layers (3) The electrospinning process for manufacturing disordered PCL / GS or PCL nanofiber interlayers is as follows: Figure 2 As shown in Figure b, the specific parameters are shown in Table 1.

[0028] (4) The manufactured electrospun film is attached to the conductive glass and fixed with the disordered side facing up on the receiving platform of the biodegradable drug-loaded meninges and PEEK skull 3D printing integrated system.

[0029] (5) Depending on the requirements for manufacturing drug-loaded and drug-free meningeal scaffolds, micron-sized PCL / nHA or PCL fibers are electrostatically printed in a controlled melt near the skull. The manufacturing process is shown in Figure 2c.

[0030] ① Fabrication of 3D-printed PCL fiber layer: PCL solid particles are loaded into a micro-syringe, and the molten electrostatically printed fibers are deposited on an electrospun film. Specific manufacturing parameters are shown in Table 2.

[0031] ② 3D printing of biodegradable nHA-loaded microfiber layers: PCL and nHA mixture solid particles are loaded into a microsyringe. Specific manufacturing parameters are shown in Table 2.

[0032] Table 2. Molten Electrostatic Printing Process Parameters for PCL and PCL / nHA Example 2: Macro- and micro-structure of a multi-level biodegradable artificial dura mater repair scaffold Figure 3 and Figure 4 The surface morphology of the PCL / GS / nHA artificial meningeal repair scaffold is shown. The surface of the PCL / GS / nHA artificial meningeal repair scaffold on the skull side is rough, while the surface of the artificial meningeal repair scaffold on the brain tissue side is smooth.

[0033] Figure 5 The SEM results show the internal multilayered microstructure of the PCL / GS / nHA artificial dura mater repair scaffold. The side view shows the three distinct layers of the PCL / GS / nHA artificial dura mater repair scaffold and the bonding between the electrospun PCL / GS fibers and the electroprinted PCL / nHA fibers. The top view reveals the uniform fiber structure of the spun and printed fibers.

[0034] Figures 6-9 The fiber diameters and orientations of electrospun ordered and disordered nanofibers are shown. The diameters of the ordered and disordered nanofibers are 520.36 ± 175.48 nm and 1662.05 ± 305.33 nm, respectively. The degrees of orientation of the ordered and disordered nanofibers within ±15° are 66.67 ± 3.17% and 25.81 ± 7.56%, respectively. These results indicate that the ordered nanofibers have better orientation.

[0035] Example 3: Drug loading and release in a multi-layered biodegradable artificial dura mater repair scaffold 1. Confirm GS successfully load-balanced. To verify whether the GS components (containing C / H / O / N / S) were successfully incorporated into the artificial meningeal repair scaffold, this embodiment employed elemental analysis, paying particular attention to changes in the content of N and S elements. Since PCL itself consists only of C, H, and O elements and contains no N or S, the presence of these two elements can serve as direct evidence of GS incorporation.

[0036] Elemental analysis allows for precise determination of the content of each element in a sample. By comparing the N and S content in PCL and PCL / GS samples, the addition of GS can be clearly identified.

[0037] The N and S contents of PCL are 0.075 ± 0.002% and 0.029 ± 0.006%, respectively, and the N and S contents of GS are 8.125 ± 0.010% and 9.315 ± 0.065%, respectively. Based on these test results and the proportion of GS, the theoretical values ​​of N and S for various artificial meningeal repair scaffolds are calculated and compared with the actual test values.

[0038] Figure 10 The nitrogen (N) content of the PCL / GS1, PCL / GS2, and PCL / GS3 artificial dura mater repair scaffolds was 0.430 ± 0.008% (theoretical value: 0.458 ± 0.002%), 0.795 ± 0.001% (theoretical value: 0.807 ± 0.003%), and 1.115 ± 0.005% (theoretical value: 1.125 ± 0.003%), respectively. The sulfur (S) content of the PCL / GS1, PCL / GS2, and PCL / GS3 artificial dura mater repair scaffolds was 0.458 ± 0.011% (theoretical value: 0.471 ± 0.009%), 0.855 ± 0.005% (theoretical value: 0.873 ± 0.011%), and 1.218 ± 0.012% (theoretical value: 1.240 ± 0.014%), respectively. The detected values ​​of N and S in each artificial dura mater repair scaffold were close to the theoretical contents, indicating successful loading of GS. Simultaneously, we observed that the elemental detection results among multiple samples in each group were similar, with very small error values. Therefore, the error bars for each group were very small. This is partly due to the relatively small amount of GS added overall, and partly suggests that the sample manufacturing process resulted in high consistency in the produced products.

[0039] 2. Confirm successful addition of nHA This embodiment uses different characterization methods (FTIR and XRD) to detect the presence of nHA in the electrostatically printed PCL / nHA artificial dura mater repair scaffold. The results are as follows: Figure 11 As shown, XRD analysis was performed on the nHA component in PCL and PCL / nHA artificial dura mater repair scaffolds. PCL showed three significant diffraction peaks at 2θ = 21.3°, 22.0°, and 23.7°, while nHA showed a significant diffraction peak at 2θ = 32°. The XRD results showed that both types of artificial dura mater repair scaffolds contained characteristic peaks of PCL, while the PCL / nHA artificial dura mater repair scaffold contained characteristic peaks of nHA, confirming the inclusion of nHA in the PCL / nHA artificial dura mater repair scaffold.

[0040] like Figure 12The results showed that the FTIR of the PCL / nHA artificial dura mater repair scaffold matched the standard infrared spectral characteristic peak PO4 of nHA. 3- Group (1040 cm) -1 The results largely overlapped, indicating that nHA was successfully added to the PCL / nHA artificial dura mater repair scaffold. The FTIR results were consistent with the XRD results, further confirming the successful addition of nHA.

[0041] like Figure 13 As shown, alizarin red staining confirmed the successful addition and uniform distribution of nHA. Alizarin red can react with Ca in nHA. 2+ The reaction produces a red complex. Alizarin Red staining was performed on the PCL and PCL / nHA artificial meningeal repair scaffolds printed by electrostatic melting on the side of the skull. The PCL / nHA artificial meningeal repair scaffold appeared red and the uniform red color indicated that nHA was successfully added and evenly distributed.

[0042] 3. Confirm the shared load of GS and nHA. EDS test results can determine the types and amounts of elements present in a sample. Figure 14 , Figure 15 and Figure 16 EDS results showed the presence and distribution of GS (S) and nHA (Ca and P) in the PCL / GS / nHA artificial meningeal repair scaffold, confirming the addition and uniform distribution of GS and nHA. Due to the large interfiber spacing on the skull side, EDS tests performed from the skull side could detect both Ca and P distributed on the PCL / nHA fibers on the skull side, indicating uniform nHA addition and showing a reticular fiber arrangement. Simultaneously, the distribution of S element on the brain tissue side could be detected through the pores between fibers, indicating uniform GS addition. However, the small interfiber spacing on the brain tissue side meant that only the distribution and presence of S element and the arrangement of ordered fibers could be detected; the presence of elements in the fibers on the skull side could not be detected through the ordered fiber layer.

[0043] 4. Analysis of Sustainable Drug Release from Bionic Artificial Meninge Repair Scaffold Regulating drug release is a key aspect of utilizing biomaterials as drug delivery carriers, enabling optimal therapeutic effects within a desired timeframe through sustained drug release. In a controlled drug release behavior assay, a biomimetic artificial dura mater repair scaffold was placed in a body temperature environment (37 °C PBS) to characterize its GS release. The long-term release of GS was validated using drug release curves.

[0044] The results are as follows Figure 17The results showed rapid drug release within 24 hours, followed by sustained release, with the cumulative release of GS increasing over time. The cumulative GS release of the drug-loaded artificial dura mater repair scaffold after 168 hours was 67.31 ± 0.75%. This initial rapid release is likely due to the presence of GS particles on the surface of the PCL / GS artificial dura mater repair scaffold, while the subsequent sustained release is due to the diffusion of GS within the PCL matrix. The GS release curve indicates that the electrospun PCL / GS artificial dura mater repair scaffold can sustainably release drugs, offering possibilities for long-term treatment.

[0045] Example 4: Mechanical property analysis of a multi-level biodegradable artificial dura mater repair scaffold For an ideal dura mater replacement, effectively preventing cerebrospinal fluid leakage is crucial. To achieve this, biomimetic artificial dura mater repair scaffolds also need to meet mechanical performance requirements. Various biomechanical properties of different samples were tested and compared.

[0046] 1. Hydrostatic pressure The resistance to hydrostatic pressure can effectively simulate the pressure that an artificial meningeal repair scaffold would withstand under intracranial pressure.

[0047] Hydrostatic pressure resistance values ​​of different samples, such as Figure 18 As shown: RD: ReDura™; P: Electrospun PCL artificial dura mater repair scaffold; PS1: Electrospun PCL / GS1 artificial dura mater repair scaffold; PS2: Electrospun PCL / GS2 artificial dura mater repair scaffold; PS3: Electrospun PCL / GS3 artificial dura mater repair scaffold; PP: Electrospun PCL + Electro-printed PCL artificial dura mater repair scaffold; PSPA: Electrospun PCL / GS2 + Electro-printed PCL / nHA artificial dura mater repair scaffold; n=3, *P<0.05, **P<0.01, nsP>0.05.

[0048] Commercially available ReDura™ (613 ± 37 mmH2O), electrospun PCL artificial dura mater repair scaffold (403 ± 7 mmH2O), PCL / GS1 artificial dura mater repair scaffold (394 ± 10 mmH2O), PCL / GS2 artificial dura mater repair scaffold (365 ± 7 mmH2O), PCL / GS3 artificial dura mater repair scaffold (346 ± 10 mmH2O), electrospun PCL + electrostatically printed PCL artificial dura mater repair scaffold (401 ± 11 mmH2O), and electrospun PCL / GS2 + electrostatically printed PCL / nHA artificial dura mater repair scaffold (i.e., PCL / GS / nHA artificial dura mater repair scaffold (375 ± 12 mmH2O) all exhibit hydrostatic pressure resistance higher than normal human intracranial pressure (95 - 204 mmH2O).

[0049] The results showed that the electrospun PCL layer near the brain tissue could withstand a decrease in hydrostatic pressure after the addition of different concentrations of gentamicin, but there was no significant difference between PCL and PCL / GS1 and PCL / GS2, but a significant difference compared to PCL / GS3. Combining the electrospun PCL layer with the molten electroprinted layer did not significantly improve the hydrostatic pressure resistance; the hydrostatic pressure resistance and leakage prevention functions were mainly undertaken by the electrospun layer. There was also no significant difference between the pure PCL three-layer membrane and the three-layer membrane with the addition of GS and nHA. All artificial meningeal repair scaffolds could withstand hydrostatic pressure greater than the normal intracranial pressure in adults and could resist cerebrospinal fluid leakage.

[0050] 2. Tensile strength and elongation at break Figure 19 and Figure 20 The tensile properties of different artificial dura mater repair scaffolds, including PCL / GS / nHA artificial dura mater repair scaffolds, are shown. P: electrospun PCL artificial dura mater repair scaffold; PS1: electrospun PCL / GS1 artificial dura mater repair scaffold; PS2: electrospun PCL / GS2 artificial dura mater repair scaffold; PS3: electrospun PCL / GS3 artificial dura mater repair scaffold; PP: electrospun PCL + electroprinted PCL artificial dura mater repair scaffold; PSPA: electrospun PCL / GS2 + electroprinted PCL / nHA artificial dura mater repair scaffold, with n=3 for each group.

[0051] Tensile stress-strain curves of different specimens are as follows: Figure 19 As shown in Figure a, the actual object used in the tensile test is as follows. Figure 19 As shown in Figure b, the tensile strength and elongation at break of each specimen are summarized in... Figure 20 .

[0052] In accordance with the YZB / National Standard 0816-2008, the mechanical requirements for biological dura mater (spinal) artificial meningeal repair scaffolds shall be that the tensile strength shall not be less than 1 MPa and the elongation at break shall not be less than 15%. The tensile strengths of the electrospun PCL artificial dura mater repair scaffolds (3.00 ± 0.40 MPa), electrospun PCL / GS1 artificial dura mater repair scaffolds (2.97 ± 0.44 MPa), electrospun PCL / GS2 artificial dura mater repair scaffolds (2.44 ± 0.31 MPa), electrospun PCL / GS3 artificial dura mater repair scaffolds (2.16 ± 0.15 MPa), electrospun PCL + electrostatically printed PCL artificial dura mater repair scaffolds (3.03 ± 0.19 MPa), and electrospun PCL / GS2 + electrostatically printed PCL / nHA artificial dura mater repair scaffolds (i.e., PCL / GS / nHA artificial dura mater repair scaffolds, 2.51 ± 0.44 MPa) are all greater than the required 1 MPa. The elongation at break of the following artificial dura mater repair scaffolds (49.98 ± 4.88%), (46.64 ± 3.16%), (46.33 ± 6.02%), (37.70 ± 3.54%), (60.56 ± 7.02%), and (44.83 ± 1.54%) all exceeded the required 15%, thus meeting the aforementioned mechanical standards. Therefore, this artificial dura mater repair scaffold is suitable for further research. The addition of GS and nHA reduced the tensile strength and elongation at break of the artificial dura mater repair scaffold to varying degrees, possibly because GS and nHA reduced the overall integrity of the polymer, and the weak bonding between PCL and bioactive particles led to a decrease in the mechanical properties of the artificial dura mater repair scaffold. The combination of the electrospun layer and the electroprinted layer improved these two parameters of the single electrospun layer. These properties allow the artificial dura mater repair scaffold to distribute operating forces to suit various injury surfaces.

[0053] 3. Bursting strength The results of the burst strength analysis are as follows Figure 21As shown, PP: electrospun PCL + electrostatically printed PCL artificial dura mater repair scaffold, i.e., PCL artificial dura mater repair scaffold; PSPA: electrospun PCL / GS2 + electrostatically printed PCL / nHA artificial dura mater repair scaffold, i.e., PCL / GS / nHA artificial dura mater repair scaffold, with n=3 for each group.

[0054] The burst strength of the PCL / GS / nHA artificial dura mater repair scaffold (2.10 ± 0.54 N) was lower than that of the PCL artificial dura mater repair scaffold (3.52 ± 0.27 N). This may be because the addition of GS and nHA nanoparticles to the PCL polymer reduces the polymer's integration, thereby lowering the scaffold's burst strength. Although the addition of GS and nHA reduces mechanical strength, they still meet the mechanical properties required for resisting cerebrospinal fluid leakage. Furthermore, the burst test curves of the three-layer artificial dura mater repair scaffold show two decreasing peaks for both scaffolds. This is because the three-layer structure, including two dense electrospun fiber layers and one fused electrostatically printed layer, collectively provides higher burst strength. The dense spun layer breaks first during the first peak decrease, while the printed layer provides secondary protection, breaking again when the second peak occurs. This further ensures the integrity of the meningeal barrier and prevents CSFL formation.

[0055] 4. Suture strength like Figure 22 As shown, PP: electrospun PCL + electrostatically printed PCL artificial dura mater repair scaffold, i.e., PCL artificial dura mater repair scaffold; PSPA: electrospun PCL / GS2 + electrostatically printed PCL / nHA artificial dura mater repair scaffold, i.e., PCL / GS / nHA artificial dura mater repair scaffold, with n=3 for each group.

[0056] The results showed that the suture strength of the PCL artificial dura mater scaffold (1.47 ± 0.26 N) was greater than that of the PCL / GS / nHA artificial dura mater scaffold (1.09 ± 0.09 N). Although the addition of GS and nHA reduced the suture strength of the PCL artificial dura mater scaffold, both the PCL and PCL / GS / nHA scaffolds still exceeded the requirement for good suture performance (1 N). These results indicate that these artificial dura mater scaffolds can be used for suturing.

[0057] 5. Bonding strength between different layers of the PCL / GS / nHA artificial meningeal repair scaffold Figure 23As shown, PS-PS: between the electrospun disordered PCL / GS layer and the ordered layer; PS-PA: between the electrospun disordered PCL / GS layer and the electro-printed PCL / nHA layer, with n=3 for each group.

[0058] The results showed that the interlayer bonding force between the two electrospun PCL / GS fibers (0.96 ± 0.07 N) was significantly higher than that between the electrospun PCL / GS fibers and the melt electroprinted PCL / nHA fibers (0.34 ± 0.13 N).

[0059] Example 5: Antibacterial Performance Analysis of Multi-Level Biodegradable Artificial Meningeal Repair Scaffold Figure 24 and Figure 25 The antibacterial properties of PCL artificial meningeal repair scaffolds with different GS concentrations against seawater bacteria were demonstrated. Among them, Figure 24 The inhibition zone (ZOI) of PCL artificial meningeal repair scaffolds with different GS concentrations was shown in seawater for 1–4 days. Figure 25 The changes in ZOI diameter of PCL artificial dura mater scaffolds at different GS concentrations from 1 to 4 days are shown. The ZOI diameters at 1 day for PCL / GS2 and PCL / GS3 were (19.47 ± 0.76 mm) and (20.47 ± 0.57 mm), respectively. Compared with the PCL artificial dura mater scaffold, the PCL / GS2 and PCL / GS3 artificial dura mater scaffolds showed significant inhibitory effects on bacteria in seawater, and the antibacterial effect increased with increasing GS concentration in the artificial dura mater scaffolds. The ZOI diameters of PCL / GS2 and PCL / GS3 artificial dura mater scaffolds at 4 days (13.74 ± 0.22 mm and 14.35 ± 0.31 mm), respectively, indicate that the antibacterial performance of the samples can be maintained for a longer period of time. Meanwhile, the PCL / GS1 artificial dura mater scaffold failed to form a significant ZOI.

[0060] SEM images such as Figure 26 As shown, various bacteria in seawater adhered extensively to the surface of the PCL artificial dura mater repair scaffold at 4 days. In contrast, the number of bacteria adhering to the PCL / GS1 artificial dura mater repair scaffold was significantly reduced, although many bacteria still remained. However, very few bacteria were observed adhering to the PCL / GS2 and PCL / GS3 artificial dura mater repair scaffolds. SEM results further confirmed the antibacterial effect of the PCL / GS2 and PCL / GS3 artificial dura mater repair scaffolds.

[0061] In summary, the PCL / GS2 and PCL / GS3 artificial meningeal repair scaffolds showed significant inhibitory activity against bacteria in seawater within 1-4 days. Meanwhile, PCL / GS1 did not form a significant zone of origin (ZOI) in seawater. The antibacterial activity of the PCL / GS2 and PCL / GS3 artificial meningeal repair scaffolds helps reduce the incidence of infection. The main bacteria in seawater are Gram-negative bacteria, including Vibrio spp., Escherichia coli spp., Salmonella spp., Proteus spp., Pseudomonas aeruginosa, Aeromonas hydrophila, and Staphylococcus aureus. Gentamicin, a water-soluble aminoglycoside antibiotic with effective broad-spectrum antibacterial activity, is often used in combination with sulfuric acid to form GS (glucose glycosides) for the treatment of Gram-negative bacterial infections. Aminoglycoside antibiotics contain multiple electrolyte groups and exhibit binding affinity to the negatively charged portions of the outer membranes of Gram-negative aerobic bacteria and nucleic acids. Electrostatic interactions increase bacterial surface permeability, allowing large amounts of GS to enter the cell and bind to bacterial 16S rRNA, ultimately leading to protein translation errors and bacterial death. The size of the zone of origin (ZOI) reflects the sensitivity of bacteria to seawater saturates (GS). Compared with the PCL artificial dura mater repair scaffold, the PCL / GS2 and PCL / GS3 artificial dura mater repair scaffolds exhibited larger ZOI diameters, which remained unchanged during bacterial culture. Therefore, the PCL / GS2 and PCL / GS3 artificial dura mater repair scaffolds can inhibit the proliferation of bacteria in seawater and are expected to exert an effective antibacterial function against tretinoinfection (TBI) under seawater immersion conditions.

[0062] Example 6: Biocompatibility Analysis of Multi-Level Biodegradable Artificial Meningeal Repair Scaffold 1. Blood compatibility Figure 27 The hemolysis rates of the PCL, PCL / GS1, PCL / GS2, and PCL / GS3 artificial dura mater repair scaffolds were shown, with quantitative values ​​of 1.29 ± 0.49%, 3.03 ± 0.23%, 3.18 ± 0.74%, and 3.88 ± 0.33%, respectively. As the GS concentration of the biomimetic artificial dura mater repair scaffold increased from GS1 to GS3, the hemolysis rate gradually increased from 3.03 ± 0.23% to 3.88 ± 0.33%, but the hemolysis rate of all samples remained below 5%, which is consistent with the implant standard. Optical images showed no signs of hemolysis in the supernatants of the various artificial dura mater repair scaffolds containing red blood cells compared to the negative control (PBS). These results indicate that the artificial dura mater repair scaffolds have good blood compatibility.

[0063] 2. Regulates the proliferation and survival of L929 fibroblasts Figure 28The proliferation results of L929 cells cultured on different types of artificial dura mater repair scaffolds in seawater are shown. Increased CCK-8 staining absorbance indicates increased cell viability; the absorbance increased to varying degrees with increasing culture time in each group. This can also reveal drug sensitivity and the rate and trend of cell proliferation. With prolonged treatment time, the OD value of cells cultured on the PCL / GS2 artificial dura mater repair scaffold significantly increased during seawater culture, indicating good biocompatibility. However, the OD value of the PCL / GS3 group was significantly lower than that of the control group. These fluctuations suggest that the PCL / GS3 artificial dura mater repair scaffold has an adverse effect on cell viability, possibly attributed to the presence of excessive GS concentration. Therefore, subsequent studies will use the concentration of PCL / GS2 as the PCL / GS concentration for the biomimetic artificial dura mater repair scaffold.

[0064] like Figure 29 and Figure 30 As shown, N+C: normal medium + control (normal culture medium without seawater + artificial meningeal scaffold); N+P: normal medium + PCL (normal culture medium without seawater + PCL + artificial meningeal scaffold); N+PS: normal medium + PCL / GS (normal culture medium without seawater + PCL / GS + artificial meningeal scaffold); S+C: seawater + control (seawater culture medium with seawater + artificial meningeal scaffold); S+P: seawater + PCL (seawater culture medium with PCL + artificial meningeal scaffold); S+PS: seawater + PCL / GS (seawater culture medium with PCL / GS + artificial meningeal scaffold); n=3 for each group.

[0065] The survival of L929 cells in seawater was assessed using dead / live cell staining. Live cells exhibited green fluorescence, while dead cells emitted a red signal. Dead / live cell staining showed that the number of cells was relatively low after 1 day of culture, with even fewer in the seawater group. After 4 days of culture, the cell number increased significantly, while the proportion of dead cells in the seawater group increased significantly. Compared to the group without the artificial meningeal repair scaffold, the proportion of dead cells was reduced in both the PCL artificial meningeal repair scaffold and the PCL / GS group. In the PCL / GS group, the proportion of live cells remained above 80% throughout the 7-day culture period, and the number of dead cells did not increase with the significant increase in live cells. The PCL / GS artificial meningeal repair scaffold showed relatively few red-stained cells after 7 days of culture, indicating a low rate of cell death.

[0066] According to GB / T 16886.5-2017 Biological Evaluation of Medical Devices Part 5: In Vitro Cytotoxicity Test Standard C.2.5, if the cell viability is less than 70%, the sample has potential cytotoxicity. Under the conditions of this test, the cell viability of 100% of the samples was greater than 70%, and no potential cytotoxicity was observed. In the seawater group, compared with the condition without the artificial meningeal repair scaffold, the ordered fibers of the PCL artificial meningeal repair scaffold provided support for cell survival in seawater, which was beneficial to cell survival, thus reducing the proportion of dead cells. The continuous release of GS in the PCL / GS artificial meningeal repair scaffold not only reduced bacteria brought by seawater, but its unique molecular structure also reduced the water contact angle, improved the hydrophilicity of the artificial meningeal repair scaffold, and made the surface of the artificial meningeal repair scaffold more conducive to the binding of cell adhesion proteins, thereby accelerating cell adhesion and spreading, and ultimately promoting cell proliferation and survival. Therefore, the biomimetic artificial meningeal repair scaffold can promote cell growth, adhesion and spreading, thereby improving cell activity and providing a safe repair platform for cell repair.

[0067] Example 7: Osteogenic Performance of Multi-Level Biodegradable Artificial Meningeal Repair Scaffold Human umbilical cord mesenchymal stem cells (HuBMSCs) have the ability to differentiate into bone tissue. Serum alkaline phosphatase (ALP) is a typical protein that runs through osteoblast differentiation and proliferation and extracellular matrix development. The early stages of osteoblast differentiation are often determined by measuring ALP activity.

[0068] Figure 31 and Figure 32 The results of ALP staining and ALP activity assays show that, compared with HuBMSCs cultured on PCL / nHA artificial dura mater repair scaffolds, HuBMSCs cultured on PCL / nHA artificial dura mater repair scaffolds exhibited more mineralized nodule formation and higher ALP activity. ALP staining was used for qualitative detection; BCIP / NBT solution formed a deep purple precipitate in the presence of alkaline phosphatase, which was visible under a microscope. Figure 31As shown in Figure 32, very little dark purple precipitate was deposited on the surface of the PCL artificial dura mater repair scaffold, while abundant dark purple precipitate was distributed on the surface of the PCL / nHA artificial dura mater repair scaffold, indicating that the introduction of nHA can promote osteogenic differentiation of HuBMSCs. Quantitative detection was performed using ALP activity assays. As shown in Figure 32, the enhanced ALP activity further indicates enhanced osteogenic differentiation of HuBMSCs on the PCL / nHA artificial dura mater repair scaffold, again verifying that the addition of nHA improves the osteogenic effect of the artificial dura mater repair scaffold. ALP can promote bone mineralization. During osteoblast differentiation and maturation, ALP activity gradually increases, contributing to bone matrix formation and calcification. ALP can also regulate cell metabolism and participate in the osteoblast response to growth factors and hormones. ALP levels are often used as one of the indicators for assessing osteogenic activity and bone metabolic status. Therefore, the qualitative and quantitative results of ALP can jointly verify the osteogenic effect of PCL / nHA.

[0069] COL-I is an important component of the bone matrix and participates in osteoblast attachment and differentiation. OCN is an osteogenic biomarker for late-stage osteoogenesis. Figure 33 , Figure 34 , Figure 35 The results of immunofluorescence staining and quantitative analysis show that, compared with the PCL artificial meningeal repair scaffold, HuBMSCs seeded on the PCL / nHA artificial meningeal repair scaffold showed increased expression of osteogenic-associated proteins COL-I and OCN after 7 and 14 days of culture, suggesting that the addition of nHA improved the osteogenic effect.

[0070] Figure 36 The PCR results showed that, compared with PCL, the mRNA levels of osteogenic-related genes OPN, OCN, COL-I, RUNX2, and ALP in HuBMSCs on the PCL / nHA artificial meningeal repair scaffold were increased to varying degrees, suggesting that the addition of nHA improved the osteogenic effect. OPN, as a structural component, participates in bone tissue construction, and RUNX2 is a key factor controlling the production of osteoblast matrix proteins. The high expression of these osteogenic markers indicates that nHA plays an osteogenic role in the PCL / nHA artificial meningeal repair scaffold, endowing it with good osteogenic properties and providing a favorable environment for osteoogenesis, which is consistent with the previous findings.

[0071] The above results all indicate that the addition of nHA can promote osteogenic differentiation. The main reason is that nHA, as a major component of human bone, can release calcium, phosphorus and other ions, increase the ion concentration in the local microenvironment, and provide a microenvironment similar to natural bone tissue for osteogenic differentiation. This is conducive to the mineralization process of osteoblasts, thereby promoting osteogenic differentiation.

[0072] Finally, it should be noted that the above description of the present invention is merely a preferred embodiment and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A multilayered degradable drug-loaded artificial dural repair stent, characterized in that, The artificial brain membrane repair bracket body is a three-layer structure, the inner layer is a directional nanofiber antibacterial layer, the middle layer is an unordered nanofiber leakage prevention layer, and the outer layer is a bone regenerative activity porous micron fiber layer.

2. The multilevel degradable drug-loaded artificial brain membrane repair stent of claim 1, wherein, The directional nanofiber antibacterial layer is highly directional electrospun polycaprolactone / kanamycin sulfate nanofiber close to the brain tissue side, which is used for simulating the single direction collagen fiber of the natural brain membrane close to the brain tissue side; the unordered nanofiber leakage prevention layer is electrospun unordered polycaprolactone / kanamycin sulfate nanofiber, which has the same fiber size as the inner layer, and is used for enhancing the mechanical properties of the brain membrane bracket; and the bone regenerative activity porous micron fiber layer is melt electrospun controllable micron polycaprolactone / nano-hydroxyapatite fiber close to the skull side.

3. The multilevel degradable drug-loaded artificial brain membrane repair stent of claim 2, wherein, The electrospun unordered polycaprolactone / kanamycin sulfate nanofiber forms an electrospun film with high compactness, prevents brain tissue adhesion, and enhances the anti-infection ability of the brain membrane bracket as an antibacterial drug carrier.

4. The multilevel degradable drug-loaded artificial brain membrane repair stent of claim 2, wherein, The micron polycaprolactone / nano-hydroxyapatite fiber comprises 12 layers of micron fibers, and the angle between adjacent two layers is 45°.

5. The multilevel degradable drug-loaded artificial brain membrane repair stent of claim 1, wherein, The thickness of the artificial brain membrane repair bracket is 296.8 μm±14.1 μm.

6. The multilevel degradable drug-loaded artificial brain membrane repair stent of claim 1, wherein, The thickness of the directional nanofiber antibacterial layer is 42.84 μm±4.37 μm, and the membrane pore size range is 1.65 μm±0.26 μm.

7. The multilevel degradable drug-loaded artificial brain membrane repair stent of claim 1, wherein, The thickness of the unordered nanofiber leakage prevention layer is 42.84 μm±4.37 μm, and the membrane pore size range is 1.02 μm±0.07 μm.

8. The multilevel degradable drug-loaded artificial brain membrane repair stent of claim 1, wherein, The thickness of the bone regenerative activity porous micron fiber layer is 211.13 μm±16.59 μm, and the membrane pore size range is 144.50 μm±19.79 μm.

9. A method for preparing a multi-layered degradable drug-loaded artificial brain membrane repair stent, characterized in that, The method comprises the following steps: (1) preparing polycaprolactone, polycaprolactone / kanamycin sulfate electrospinning solution and polycaprolactone, polycaprolactone / nano-hydroxyapatite electrospinning particles, which comprises: ① polycaprolactone spinning solution: preparing a polycaprolactone spinning solution with a content of 15 w / v% by using HFIP as a solvent; ② polycaprolactone / kanamycin sulfate electrospinning solution: using HFIP as a solvent, adding kanamycin sulfate first, then adding polycaprolactone particles by using a magnetic stirrer and an ultrasonic cleaning machine to uniformly disperse the kanamycin sulfate, and finally the concentration of polycaprolactone and kanamycin sulfate in the polycaprolactone / kanamycin sulfate electrospinning solution is 15 w / v% and 1.5 w / v% respectively; ③ polycaprolactone electrospinning particles: selecting biological-grade polycaprolactone particles; ④ polycaprolactone / nano-hydroxyapatite electrospinning particles: using HFIP as a solvent and a magnetic stirrer to completely dissolve and uniformly disperse the first-added nano-hydroxyapatite particles, then adding polycaprolactone particles, again uniformly dispersing, and then completely volatilizing HFIP, cutting the polycaprolactone / nano-hydroxyapatite film into polycaprolactone / nano-hydroxyapatite electrospinning particles, wherein the mass ratio of nano-hydroxyapatite particles to polycaprolactone particles is 1:

10. (2) The prepared polycaprolactone / kanamycin sulfate electrospinning solution is loaded into a syringe and clamped on a single-channel precision syringe pump, a layer of tin paper is pasted on the drum receiving device to facilitate collection, the syringe needle tip is connected to the positive electrode, the drum receiving device is grounded, the parameters of electrospinning are set, the voltage is 13 kV, the flow rate is 1.2 mL / h, the receiving distance is 11 mm, the rotating speed is 3600 r / min, and the electrospinning is performed for 30 min; (3) Adjust the electrospinning parameter rotating speed to 120 r / min, set the electrospinning parameter voltage to 13 kV, flow rate to 1.2 mL / h, and receiving distance to 11 mm, and continue electrospinning for 90 min; (4) Stop electrospinning, place the obtained film in a fume hood to fully evaporate the solvent, then cut it into a size of 50 mm*50 mm and paste it on a glass, and then fix it on a moving receiving platform of a melt electrostatic printing device; (5) The polycaprolactone / nano-hydroxyapatite solid particles are loaded into a micro-syringe and clamped on a high-precision syringe pump, the heating switch is turned on, the heating temperature is set to 85℃, after the particles are melted, the syringe pump control software is turned on, the flow rate is set to 30 μL / h, the receiving distance is adjusted to 5 mm after the flow rate is stabilized, the high-voltage power supply is adjusted to 5 kV, and after a Taylor cone is formed at the needle tip, the three-dimensional platform moves according to the pre-programmed program, the angle of each layer of fibers changes by 45 degrees, a total of 12 layers, and the melt electrostatic printing fibers are deposited on the electrospun film to obtain a degradable drug-loaded artificial brain membrane repair scaffold.

10. Use of the multi-tiered degradable drug-loaded artificial brain membrane repair stent according to claim 1 in bio-tissue engineering. The multi-level degradable drug-loaded artificial brain membrane repair scaffold is integrally formed by 3D printing, polycaprolactone fibers are added with kanamycin sulfate and nano-hydroxyapatite to manufacture a degradable drug-loaded brain membrane scaffold with a three-layer structure, which is used to realize sustainable release of drugs.

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