An annulus fibrosus repair brace and method of making the same

Abstract

The application discloses a kind of annulus fibrosus repair bracket and its preparation method, it is related to medical instrument field, it includes at least two layers of nanofiber membrane, the nanofiber in the nanofiber membrane is oriented arrangement, the nanofiber in the adjacent two layers of the nanofiber membrane is arranged in the direction of orientation crossing, the nanofiber membrane is made by the crosslinking after composite spinning solution directional electrospinning, the composite spinning solution includes fibroin, polyvinyl alcohol, chondroitin sulfate and solvent;The weight ratio of the fibroin and the polyvinyl alcohol is 1:0.4-2.5.This application is conducive to maintaining the cell morphology and spreading state of annulus fibrosus cell and improving proliferation level on one hand;Improve anti-inflammatory effect;It is conducive to improving the matrix metabolic imbalance of annulus fibrosus cell under inflammatory microenvironment.On the other hand, it is conducive to maintaining intervertebral disc height, improving MRI signal, promoting lamina reconstruction and weakening local inflammation.

Description

Technical Field

[0001] This application relates to the field of medical devices, and in particular to a fibrous annulus repair scaffold and its preparation method. Background Technology

[0002] The occurrence of intervertebral disc degenerative diseases is closely related to the destruction of the annulus fibrosus structure. The integrity of the annulus fibrosus plays an important role in maintaining intradiscal pressure and protecting the nucleus pulposus. Therefore, repairing annulus fibrosus damage is a key step in the treatment of intervertebral disc degeneration. Currently, clinical treatment mainly focuses on conservative or surgical treatment, but neither can effectively restore the structure and function of the annulus fibrosus.

[0003] The development of tissue engineering technology has provided new ideas for annulus fibrosus repair. Currently, 3D printing technology is being used to model the annulus fibrosus, and polycaprolactone and glacial acetic acid are being used to 3D print pores with a diameter of 50×50-100×100μm. 2 The fiber ring stacked scaffold with a fiber diameter of 20-50μm and 6 layers has a mesh-like interlaced structure. Although it improves the physical load-bearing capacity of the fiber ring, it has limited biological effects on the orientation growth of fiber ring cells and the promotion of matrix repair. Summary of the Invention

[0004] In view of the shortcomings of the above-mentioned related technologies, this application provides a fiber ring repair scaffold and its preparation method.

[0005] Firstly, the fiber annulus repair scaffold provided in this application adopts the following technical solution: A fibrous ring repair scaffold includes at least two nanofiber membranes, wherein the nanofibers in the nanofiber membranes are oriented in an oriented manner, and the orientation directions of the nanofibers in adjacent nanofiber membranes are intersected. The nanofiber membranes are obtained by cross-linking after directional electrospinning with a composite spinning solution, wherein the composite spinning solution includes silk fibroin, polyvinyl alcohol, chondroitin sulfate and a solvent; the weight ratio of the silk fibroin to the polyvinyl alcohol is 1:0.4-2.5.

[0006] Preferably, the weight ratio of the silk fibroin to the polyvinyl alcohol is 1:0.8-1.2.

[0007] Preferably, the weight ratio of the silk fibroin to the chondroitin sulfate is 1:0.05-0.2.

[0008] Preferably, the weight ratio of the silk fibroin to the chondroitin sulfate is 1:0.05-0.15.

[0009] Preferably, the weight ratio of the silk fibroin to the chondroitin sulfate is 1:0.08-0.12.

[0010] Preferably, the weight ratio of the silk fibroin to the chondroitin sulfate is 1:0.1.

[0011] Preferably, the weight ratio of the silk fibroin to the solvent is 0.3-0.7:1.

[0012] Preferably, the average diameter of the nanofibers in the nanofiber membrane is 400-700 nm.

[0013] Preferably, the preparation of the silk fibroin includes the following steps: taking silkworm cocoons, heating and degumming them under alkaline conditions, dissolving them in a lithium bromide solution to obtain a mixed solution, dialysis the mixed solution to remove lithium bromide to obtain a silk fibroin solution, and freeze-drying the silk fibroin solution to obtain silk fibroin.

[0014] Preferably, the included angle between the orientation directions of the nanofibers in the two adjacent nanofiber membranes is 20°-40°.

[0015] Preferably, the directional electrospinning involves controlling the spinneret direction to be perpendicular to the radial direction of the roller collector, maintaining a constant rotation speed of the roller collector, and achieving directional alignment of nanofibers.

[0016] Preferably, the parameters of the directional electrospinning are: voltage 15-17kV, spinneret liquid output speed 0.4-0.6mL / h, receiving distance 17-19cm, and rotation speed 1200-1400r / min.

[0017] Secondly, the method for preparing a fiber annulus repair scaffold provided in this application adopts the following technical solution: A method for preparing a fibrous annulus repair scaffold includes the following steps: dissolving polyvinyl alcohol and silk fibroin in a solvent to obtain a polyvinyl alcohol solution and a silk fibroin solution, mixing and stirring the polyvinyl alcohol solution, silk fibroin solution and chondroitin sulfate evenly to obtain a composite spinning solution; performing directional electrospinning on the composite spinning solution to obtain an electrospun membrane; performing cross-linking treatment on the electrospun membrane to obtain a single-layer nanofiber membrane; and stacking multiple nanofiber membranes in a cross-shaped manner with their fiber orientation directions to obtain the fibrous annulus repair scaffold.

[0018] Preferably, the crosslinking treatment includes treatment with ethanol for 5-30 minutes and treatment with glutaraldehyde for 1-12 hours.

[0019] In summary, this application includes at least one of the following beneficial technical effects: This invention utilizes directional electrospinning of nanofibers, primarily composed of silk fibroin, polyvinyl alcohol, and chondroitin sulfate, to successfully construct nanofiber membranes with oriented nanofibers. These membranes are then stacked in a cross-sectional pattern along the fiber orientation to create multilayer nanofiber membranes that mimic the annulus fibrosus (Annulus fibrosus) structure, forming a multilayered cross-linked scaffold for annulus fibrosus repair. On one hand, in in vitro experiments, guiding the directional growth of annulus fibrosus cells helps maintain their morphology and spread, and increases their proliferation; it also enhances the anti-inflammatory effect of the annulus fibrosus repair scaffold under inflammatory conditions and helps improve the metabolic imbalance of the annulus fibrosus cells in the inflammatory microenvironment. On the other hand, in in vivo experiments, it helps maintain intervertebral disc height, improves MRI signals, promotes laminar remodeling, and reduces local inflammation. Attached Figure Description

[0020] Figure 1 The images are scanning electron microscope (SEM) images of nanofiber membranes prepared using the method described in Comparative Example 1 with different ratios of polyvinyl alcohol and silk fibroin.

[0021] Figure 2 The results show the diameter analysis of nanofibers in nanofiber membranes prepared using the preparation method of Comparative Example 1 with different ratios of polyvinyl alcohol and silk fibroin raw materials. Figure 3 These are scanning electron microscope images of the nanofiber membranes obtained during the preparation processes of Examples 1-3, Comparative Example 1, and Comparative Example 2. Figure 4 These are comparison images of the Fourier transform infrared spectra of the nanofiber membranes obtained during the preparation processes of Examples 1-3 and Comparative Example 2. Figure 5 These are comparison diagrams of the tensile stress of the nanofiber membranes obtained during the preparation process of Examples 1-3 and Comparative Example 2; Figure 6 These are scanning electron microscope (SEM) comparison images of the cell growth status on the nanofiber membranes prepared in Example 1 and Comparative Example 2 after cell compatibility and adhesion experiments. Figure 7 The results of live / dead cell staining after cell compatibility and adhesion experiments on the nanofiber membranes prepared in Examples 1-3 and Comparative Example 1; Figure 8 The results of phalloidin staining were obtained after cell compatibility and adhesion experiments on the nanofiber membranes prepared in Examples 1-3 and Comparative Example 1. Figure 9 The results of CCK-8 cell proliferation experiments were performed on the nanofiber membranes prepared in Examples 1-3 and Comparative Example 1 after conducting cell compatibility and adhesion experiments. Figure 10This is a comparison of the mRNA expression results of inflammation-related genes Il6, Tnf, Ptgs2, and Nos2 when annulus fibrosus cells after inflammatory stimulation were co-cultured with nanofiber membranes prepared in Examples 1-2 and Comparative Example 1, respectively. Figure 11 This is a comparison of the protein expression results of TNF-α and COX-2 when annulus fibrosus cells after inflammatory stimulation are co-cultured with nanofiber membranes prepared in Examples 1-2 and Comparative Example 1, respectively. Figure 12 This is a comparison of the relative protein expression levels of TNF-α and COX-2 when annulus fibrosus cells after inflammatory stimulation are co-cultured with nanofiber membranes prepared in Examples 1-2 and Comparative Example 1, respectively. Figure 13 The image shows a comparison of the immunofluorescence signals of TNF-α and COX-2 when annulus fibrosus cells after inflammatory stimulation were co-cultured with nanofiber membranes prepared in Examples 1-2 and Comparative Example 1, respectively. Figure 14 This is a comparison of the relative fluorescence intensities of TNF-α and COX-2 when annulus fibrosus cells after inflammatory stimulation are co-cultured with nanofiber membranes prepared in Examples 1-2 and Comparative Example 1, respectively. Figure 15 This is a comparison of the mRNA expression results of Col1a1, Col2a1, Acan, Mmp3, and Mmp13 when annulus fibrosus cells after inflammatory stimulation were co-cultured with nanofiber membranes prepared in Examples 1-2 and Comparative Example 1, respectively. Figure 16 The graph shows a comparison of the expression results of COL I, COL II and MMP13 proteins when annulus fibrosus cells after inflammatory stimulation are co-cultured with nanofiber membranes prepared in Examples 1-2 and Comparative Example 1, respectively. Figure 17 This is a comparison of the relative protein expression levels of COL I, COL II, and MMP13 when annulus fibrosus cells after inflammatory stimulation are co-cultured with nanofiber membranes prepared in Examples 1-2 and Comparative Example 1, respectively. Figure 18 The image shows a comparison of the immunofluorescence signals of COL I, COL II, and MMP13 when annulus fibrosus cells after inflammatory stimulation were co-cultured with nanofiber membranes prepared in Examples 1-2 and Comparative Example 1, respectively. Figure 19 This is a comparison of the relative fluorescence intensities of COL I, COL II, and MMP13 when annulus fibrosus cells after inflammatory stimulation are co-cultured with nanofiber membranes prepared in Examples 1-2 and Comparative Example 1, respectively. Figure 20 The results of X-ray examination of the intervertebral discs during in vitro experiments using the annulus fibrosus repair scaffold of Example 1 and the annulus fibrosus scaffold of Comparative Example 1. Figure 21 The intervertebral disc height index is the intervertebral disc height when the annulus fibrosus repair scaffold of Example 1 and the annulus fibrosus scaffold of Comparative Example 1 are used in an in vitro experiment. Figure 22 These are transverse images of the intervertebral discs during in vitro experiments using the annulus fibrosus repair scaffold of Example 1 and the annulus fibrosus scaffold of Comparative Example 1. Figure 23 These are sagittal T2-weighted images of the intervertebral discs during in vitro experiments using the annulus fibrosus repair scaffold of Example 1 and the annulus fibrosus scaffold of Comparative Example 1. Figure 24 The results of quantitative analysis of gray values ​​and Pfirrmann classification in sagittal T2-weighted images during in vitro experiments using the annulus fibrosus repair scaffold of Example 1 and the annulus fibrosus scaffold of Comparative Example 1 are as follows: Figure 25 The staining results of hematoxylin-eosin on the intervertebral discs were obtained during in vitro experiments using the annulus fibrosus repair scaffold of Example 1 and the annulus fibrosus scaffold of Comparative Example 1. Figure 26 The staining results of Safranin O-Fix Green on the intervertebral discs during in vitro experiments using the annulus fibrosus repair scaffold of Example 1 and the annulus fibrosus scaffold of Comparative Example 1. Figure 27 The results of masson staining of the intervertebral disc were obtained when the annulus fibrosus repair scaffold of Example 1 and the annulus fibrosus scaffold of Comparative Example 1 were used in in vitro experiments. Figure 28 The results of COL I expression in the intervertebral disc during in vitro experiments using the annulus fibrosus repair scaffold of Example 1 and the annulus fibrosus scaffold of Comparative Example 1. Figure 29 The results of Aggrecan expression in the intervertebral disc were obtained when the annulus fibrosus repair scaffold of Example 1 and the annulus fibrosus scaffold of Comparative Example 1 were used in in vitro experiments. Figure 30 The results show the expression of IL-1β in the intervertebral disc during in vitro experiments using the annulus fibrosus repair scaffold of Example 1 and the annulus fibrosus scaffold of Comparative Example 1. Detailed Implementation

[0022] The present application will be further described in detail below with reference to the embodiments. The following embodiments are for illustrative purposes only and should not be considered as limiting the scope of the invention. Unless otherwise specified, specific conditions in the following embodiments were performed under conventional conditions or conditions recommended by the manufacturer. Unless otherwise specified, the methods used are conventional methods known in the art, and the consumables and reagents used are commercially available. Unless otherwise stated, the technical and scientific terms used herein have the same meaning as those familiar to those skilled in the art. Furthermore, any methods or materials similar to or equivalent to those described herein may also be applied to the present invention.

[0023] Example 1 Example 1 of this application provides a fiber annulus repair scaffold, the specific steps of which are as follows: For the extraction of silk fibroin, 10g of silkworm cocoons were added to 4L of sodium carbonate solution (0.02M) and boiled for 30min to remove sericin components, resulting in degummed silkworm cocoons. The degummed silkworm cocoons were then placed in lithium bromide solution (9.3M) at 60℃ for 4h to dissolve, resulting in a mixed solution. The mixed solution was then placed in a dialysis bag and dialyzed for 4 days, with the water changed twice a day, to remove lithium bromide and obtain a silk fibroin solution. The silk fibroin solution was then freeze-dried to obtain purified silk fibroin, which was stored at 4℃ for later use.

[0024] To prepare the composite spinning solution, 5g of polyvinyl alcohol granules were added to 50mL of deionized water and stirred and heated in a 90℃ water bath for more than 2 hours to prepare a 10% (w / v) polyvinyl alcohol solution. The solution was then cooled to room temperature for later use. 3g of silk fibroin was added to 30mL of deionized water and magnetically stirred at room temperature for 12 hours to prepare a 10% (w / v) silk fibroin solution. 5mL of the polyvinyl alcohol solution and 5mL of the silk fibroin solution were mixed, and 0.05g of chondroitin sulfate was added. The mixture was stirred at room temperature for 2 hours to obtain the composite spinning solution.

[0025] To prepare the nanofiber membrane, the composite spinning solution was loaded into a 5 mL syringe, connected to a stainless steel needle with an inner diameter of 0.6 mm, and installed in an electrospinning device. The needle was positioned towards the roller collector, and the spinning direction was perpendicular to the radial direction of the roller collector. Spinning was carried out for 4 hours. The electrospinning parameters were set as follows: voltage 16 kV, feed speed 0.5 mL / h, receiving distance 18 cm, and roller speed 1300 r / min. The ambient temperature was controlled at 27℃, and the relative humidity was controlled below 20%. The electrospun membrane was obtained and immersed in anhydrous ethanol for 20 min. Then, it was placed in a fume hood to allow the ethanol to evaporate completely. Then, it was transferred and suspended in a sealed container containing 20 mL of glutaraldehyde aqueous solution (20% v / v). Crosslinking was carried out at room temperature for 5 h. After crosslinking, it was dried in a 50℃ oven for 2 h to remove residual glutaraldehyde, thus obtaining the nanofiber membrane.

[0026] The fabrication of the annulus fibrosus repair scaffold involves cutting a nanofiber membrane into multiple uniformly sized sheets. The size and number of sheets are determined based on the shape and size of the annulus fibrosus defect, and the fiber orientation direction is marked on each sheet. Using the fiber orientation direction of the first nanofiber membrane as a reference direction, the second nanofiber membrane is rotated 30° relative to the first layer and then stacked. Each subsequent layer is added after rotating 30° relative to the fiber orientation direction of the previous layer. In this embodiment, a total of 40 layers are stacked, forming a multilayered structure with adjacent layers having a 30° angle between their fiber orientation directions. During stacking, the edges of each membrane layer are kept aligned, and molds, positioning plates, or clamping fixtures can be used to limit and fix each membrane layer to prevent interlayer displacement, resulting in an annulus fibrosus repair scaffold with a 30° multilayered staggered orientation structure.

[0027] Example 2 Example 2 of this application provides a fiber ring repair scaffold. The difference between Example 2 and Example 1 is that the amount of chondroitin sulfate added in the preparation step of the composite spinning solution in Example 2 is 0.025g.

[0028] Example 3 Example 3 of this application provides a fiber ring repair scaffold. The difference between Example 3 and Example 1 is that the amount of chondroitin sulfate added in the preparation step of the composite spinning solution in Example 3 is 0.075g.

[0029] Comparative Example 1 Comparative Example 1 of this application provides a fiber ring scaffold. The difference between Comparative Example 1 and Example 1 is that chondroitin sulfate is not added in the preparation step of the composite spinning solution of Comparative Example 1.

[0030] Comparative Example 2 Comparative Example 2 of this application provides a fiber ring scaffold. The difference between Comparative Example 2 and Example 1 is that the amount of chondroitin sulfate added in the preparation step of the composite spinning solution of Comparative Example 1 is 0.1g.

[0031] Testing and Inspection (1) Nanofiber membranes were prepared using composite spinning solutions with different ratios of polyvinyl alcohol and silk fibroin according to the preparation method of Comparative Example 1. The film-forming properties of composite spinning solutions with different ratios of polyvinyl alcohol and silk fibroin were studied. Five groups were set up with polyvinyl alcohol and silk fibroin weight ratios of 1:9, 3:7, 5:5, 7:3 and 9:1, labeled as P1S9, P3S7, P5S5, P7S3 and P9S1, respectively. Among them, the polyvinyl alcohol and silk fibroin weight ratio of 5:5 was Comparative Example 1. The scanning electron microscope images of the nanofiber membranes labeled as P1S9, P3S7, P5S5, P7S3 and P9S1 are shown below. Figure 1As shown in the figure. The results showed that the P1S9 group had poor spinnability and disordered fiber arrangement with no obvious directional structure due to the high proportion of silk fibroin. As the proportion of polyvinyl alcohol increased, the fiber continuity and orientation improved. The P5S5 group showed smooth fiber surface, neat arrangement, and uniform diameter distribution, with both good spinnability and structural stability. After further increasing the proportion of polyvinyl alcohol to P7S3 and P9S7, the directional structure was destroyed and the fiber arrangement became disordered again.

[0032] The nanofiber diameters of nanofiber membranes labeled P1S9, P3S7, P5S5, P7S3, and P9S1 were analyzed using ImageJ image analysis software. The results are as follows: Figure 2 As shown in the figure, fiber diameter analysis revealed that the average diameter increased from approximately 324 nm to 622 nm with increasing polyvinyl alcohol (PVA) content. In summary, a 5:5 weight ratio of PVA to silk fibroin is preferred for preparing nanofiber membranes, as it improves the fiber-forming properties, continuity, and orientation of the nanofiber membranes.

[0033] (2) Scanning electron microscope images of the nanofiber membranes obtained in the preparation processes of Examples 1-3, Comparative Example 1 and Comparative Example 2 are shown below. Figure 3 As shown, they are sequentially labeled P5S5C 10 P5S5C5, P5S5C 15 P5S5 and P5S5C 20 Fourier transform infrared spectrum comparison diagram as follows Figure 4 As shown in the scanning electron microscope (SEM) images, the nanofibers in the P5S5C5 group are significantly thinner and their orientation is slightly reduced; P5S5C 10 The group maintains good continuity and orientation, the fibers are uniform, and there are no obvious beading or breakage on the surface. Fiber diameter analysis yielded P5S5C. 10 The average diameter of the nanofibers in the group was 543.87 ± 97.46 nm, balancing uniformity and structural stability. With further increases in chondroitin sulfate content, the interweaving of fibers in the nanofiber membrane increased, while orientation decreased. Fourier transform infrared spectroscopy confirmed that the addition of chondroitin sulfate resulted in the appearance or enhancement of chondroitin sulfate-related characteristic absorption peaks in the nanofiber membrane, indicating successful incorporation of chondroitin sulfate into the nanofiber membrane system.

[0034] Mechanical tests were performed on the nanofiber membranes obtained during the preparation processes of Examples 1-3 and Comparative Example 2, and the tensile stress comparison diagrams are shown below. Figure 5 As shown, they are sequentially labeled P5S5C 10 P5S5C5, P5S5C 15 and P5S5C 20 The results show that P5S5C 10The group exhibited the highest tensile stress, reaching 3.5 MPa, significantly superior to P5S5C5 and P5S5C. 15 and P5S5C 20 In summary, a 5:5 weight ratio of polyvinyl alcohol to silk fibroin is preferred for preparing nanofiber membranes, and the chondroitin sulfate content should be 10% of the total mass of polyvinyl alcohol and silk fibroin, which is beneficial to improving the orientation and mechanical properties of the nanofiber membranes.

[0035] (3) In vitro experiments: 1. Cell compatibility and adhesion experiments: Primary rat annulus fibrosus cells were cultured in vitro, and cells from passages 3-5 were used for the experiments. The nanofiber membranes prepared in Examples 1-3 and Comparative Examples 1-2 were cut into 10 mm diameter discs, sterilized with 75% ethanol, and irradiated with UV light for at least 30 min before being placed in 24-well plates and labeled sequentially as P5S5C. 10 P5S5C5, P5S5C 15 P5S5 and P5S5C 20 Subsequently, the annulus fibrosus cells were divided into groups of 5 × 10⁻⁶. 4 The nanofiber membranes were seeded at a density of 1 scaffold per nanofiber and incubated at 37°C in a 5% CO2 incubator. 20 Group and P5S5C 10 Scanning electron microscopy comparison images of the growth status of fibrous ring cells on nanofiber membranes in the group, as shown below. Figure 6 As shown. The results show that P5S5C 10 In the group, the annulus fibrosus cells are elongated on the surface of oriented fibers and extend along the fiber orientation direction, while in non-orientation cells such as P5S5C... 20 The random spreading of the nanofiber membranes in the group indicates that the nanofiber membranes used in Example 1 are beneficial for promoting the directional growth of annulus fibrosus cells.

[0036] Live / dead cell staining: After culturing for 24 h, cells on the nanofiber membranes of Examples 1-3 and Comparative Example 1 were gently washed twice with PBS, and then incubated with Calcein-AM / PI staining solution (Calcein-AM 2 μM, PI 4.5 μM) for 20 min at 37°C in the dark. The green (live cells) and red (dead cells) signals were then observed using a fluorescence microscope to evaluate cell viability. The results are as follows: Figure 7 As shown in the figure. The results showed that a large number of green live cells were visible on the surface of each group, with almost no obvious red dead cells, indicating that the nanofiber membranes of each group had no significant cytotoxicity.

[0037] Phalloidin staining: After culturing for 24 h, the nanofiber membranes from Examples 1-3 and Comparative Example 1 were gently washed with PBS, fixed in 4% paraformaldehyde for 15 min, perforated with 0.1% Triton X-100 for 10 min, and blocked with 5% BSA for 1 h. iF488-Phalloidin (1:200) was added and incubated for 1 h, followed by washing with PBS three times, DAPI staining for 5 min, mounting with anti-fluorescence attenuation mounting solution, and observation of cytoskeleton arrangement and spreading under a fluorescence microscope. The results are as follows: Figure 8 As shown. The results show that P5S5C 10 In this group, the fibrous ring cells are arranged regularly along the fiber orientation direction, exhibiting the most obvious directional growth state.

[0038] CCK-8 cell proliferation assay: The nanofiber membranes from Examples 1-3 and Comparative Example 1 were removed on days 1, 3, and 5 of culture and added to culture medium containing 10% CCK-8 reagent (total volume 200 μL / well), and incubated at 37°C for 2 h. The optical density (OD value) was measured at 450 nm using a microplate reader to reflect cell proliferation on the scaffolds in each group. At least three replicates were performed for each group. The average value and standard deviation were calculated. The results are shown below. Figure 9 As shown in the figure. The results showed that the OD450 values ​​of each group increased with the extension of culture time, among which P5S5C... 10 The group had the highest proliferation level.

[0039] In summary, when preparing nanofiber membranes, a weight ratio of 5:5 for polyvinyl alcohol and silk fibroin is preferred, and the amount of chondroitin sulfate is 10% of the total mass of polyvinyl alcohol and silk fibroin. This not only helps maintain the annulus fibrosus cells but also promotes the directional growth of the annulus fibrosus cells and increases their proliferation level.

[0040] 2. In vitro anti-inflammatory experiment: Annulus fibrosus cells (labeled con) were used as the control group. An inflammatory microenvironment model was established by stimulating Annulus fibrosus cells with 10 ng / mL IL-1β for 24 h. Subsequently, the inflammatory-stimulated Annulus fibrosus cells were co-cultured with nanofiber membranes of different experimental groups for 48 h. The groups included the IL-1β group (without nanofiber membrane), the IL-1β+P5S5 group (with nanofiber membrane prepared in Comparative Example 1), the IL-1β+P5S5C5 group (with nanofiber membrane prepared in Example 2), and the IL-1β+P5S5C5 group. 10 The experiment consisted of four groups (each with the nanofiber membrane prepared in Example 1 added). The mRNA expression of inflammation-related genes Il6, Tnf, Ptgs2, and Nos2 was detected in each group, and the results are shown below. Figure 10 As shown; the results of detecting TNF-α and COX-2 protein expression in each group are as follows. Figure 11 As shown, the relative protein expression levels are as follows: Figure 12 As shown; the immunofluorescence signals of TNF-α and COX-2 in each group were detected, and the results are as follows. Figure 13 As shown, the relative fluorescence intensity results are as follows: Figure 14 As shown in the figure. The results showed that, compared with the control group, the expression levels of inflammation-related genes and protein levels were significantly increased in the experimental group. Compared with the IL-1β group, the IL-1β+P5S5 group, IL-1β+P5S5C5 group, and IL-1β+P5S5C5 group showed significantly higher levels of expression. 10 The expression levels and protein levels of inflammation-related genes were downregulated in the group, especially IL-1β+P5S5C. 10 The results of Western blotting and immunofluorescence in the group were consistent, showing the most significant inhibition of TNF-α and COX-2 expression, which was close to the state of the control group. This indicates that when preparing nanofiber membranes, the preferred weight ratio of polyvinyl alcohol and silk fibroin is 5:5, and the chondroitin sulfate content is 10% of the total mass of polyvinyl alcohol and silk fibroin, which is beneficial to improving the anti-inflammatory ability of nanofiber membranes.

[0041] 3. Extracellular matrix metabolic regulation experiment: Based on the above in vitro anti-inflammatory experiments, the mRNA expression of Col1a1, Col2a1, Acan, Mmp3, and Mmp13 in each group was further detected. The results are as follows: Figure 15 As shown; the results of detecting the expression of COL I, COL II, and MMP13 proteins in each group are as follows. Figure 16 As shown, the relative protein expression levels are as follows: Figure 17 As shown; the immunofluorescence signals of COL I, COL II, and MMP13 in each group were detected, and the results are as follows. Figure 18 As shown, the relative fluorescence intensity results are as follows: Figure 19 As shown in the figure. The results showed that IL-1β stimulation led to a decrease in the expression of synthesis-related genes Col1a1, Col2a1, and Acan, while the expression of degradation-related genes Mmp3 and Mmp13 increased. The IL-1β+P5S5 group showed a partial inhibitory effect on degradation-related genes, but the improvement was limited; IL-1β+P5S5C 10 The group significantly increased the expression of synthesis-related genes and inhibited the expression of degradation-related genes. The protein levels showed the same trend, indicating that the preferred weight ratio of polyvinyl alcohol and silk fibroin to prepare nanofiber membranes is 5:5, and the chondroitin sulfate content is 10% of the total mass of polyvinyl alcohol and silk fibroin, which is beneficial to improving the matrix metabolic imbalance of annulus fibrosus cells in the inflammatory microenvironment and providing a favorable environment for annulus fibrosus repair.

[0042] (4) In vivo experiments: Adult SD rats were selected as experimental animals and divided into four groups: sham-operated group (labeled Sham), model group (labeled Con), P5S5 group (labeled P5S5C0), and P5S5C... 10 Group (labeled P5S5C) 10 In the sham surgery group, only the intervertebral disc was exposed, without annulus fibrosus injury or IL-1β injection; in the model group, annulus fibrosus defects were created and IL-1β was injected, but no scaffold was implanted. The model group was constructed as follows: after anesthesia, the L3 / 4 and L4 / 5 intervertebral discs of the lumbar spine were exposed via a ventral approach. A defect of 2 mm × 1 mm × 1 mm in size was created in the annulus fibrosus using a microscalpel, and 100 ng of IL-1β was injected into the defect site to establish an annulus fibrosus injury combined with inflammation-induced intervertebral disc degeneration model; in the P5S5 group, the annulus fibrosus scaffold of Comparative Example 1 was implanted in the annulus fibrosus defect site of the model of annulus fibrosus injury combined with inflammation-induced intervertebral disc degeneration; P5S5C 10 In one group, the annulus fibrosus repair scaffold of Example 1 was implanted into the annulus fibrosus defect site in a model of intervertebral disc degeneration induced by combined annulus fibrosus injury and inflammation. Subsequently, the annulus fibrosus repair scaffolds of different groups were cut to a size matching the annulus fibrosus defect and immediately implanted into the defect site. Imaging and histological examinations were performed at weeks 2 and 8 postoperatively.

[0043] X-rays were used to detect changes in intervertebral disc height at 2 and 8 weeks post-surgery, and the results were as follows: Figure 20 As shown, the intervertebral disc height index was calculated, and the results are as follows. Figure 21 As shown in the figure. The results showed that the model group exhibited a significant decrease in the intervertebral disc height index, while the P5S5C using the annulus fibrosus repair scaffold of Example 1... 10 The intervertebral disc height index of the group was the smallest, and the effect was significantly better than that of the P5S5 group using the annulus fibrosus scaffold of Comparative Example 1. This indicates that the annulus fibrosus repair scaffold of Example 1 is beneficial to reduce the degree of decrease in intervertebral disc height index in the intervertebral disc degeneration model induced by annulus fibrosus injury combined with inflammation.

[0044] Magnetic resonance imaging was used to detect the hydration status and tissue integrity of the intervertebral disc. Transverse images are shown below. Figure 22 As shown. The results indicate that the signal in the nucleus pulposus region of the model group was significantly weakened or even disappeared, the structural boundaries were blurred, and P5S5C... 10 The nucleus pulposus structure of the P5S5 group and the P5S5 group remained intact, especially the P5S5C group. 10 The integrity of the nucleus pulposus structure in the group was closest to that in the sham surgery group.

[0045] Further analysis of sagittal T2-weighted images, such as Figure 23 As shown, the results of grayscale value quantification and Pfirrmann classification are as follows: Figure 24As shown in the image. The results show that the model group exhibited low signal intensity and intervertebral space collapse in the images, while P5S5C... 10 The signal enhancement of the group, the good morphology of the intervertebral disc, and the quantitative gray value and Pfirrmann classification results further validated P5S5C. 10 The group with the lowest degree of degeneration and the best repair effect indicates that the annulus fibrosus repair scaffold of Example 1 is beneficial to inhibiting intervertebral disc degeneration and promoting annulus fibrosus repair.

[0046] After the examination, the corresponding spinal segments were taken, fixed, decalcified, embedded in paraffin, and sectioned to a thickness of 5 μm. Hematoxylin-eosin (HE), safranin O-fast green, and Masson staining were then performed, with results as follows: Figure 25-27 As shown, the structure of the annulus fibrosus lamina, the content of proteoglycans in the nucleus pulposus, and the arrangement of collagen fibers in the annulus fibrosus were observed. Hematoxylin-eosin staining results indicated that the model group showed annulus fibrosus lamina breakage, disordered arrangement, and inflammatory cell infiltration at 2 weeks, which worsened further at 8 weeks; P5S5C 10 The P5S5 group and the P5S5 group showed different degrees of structural improvement, with P5S5C showing the most significant improvement. 10 At 8 weeks, the annulus fibrosus structure in the model group was more complete, and the annulus fibrosus lamellae were more regularly arranged, similar to the sham-operated group with a clear annulus fibrosus lamellae arrangement; Safranin O-Fixed Green staining showed that the model group had significant proteoglycan loss, P5S5C 10 The enhanced safranin staining in groups P5S5 and P5S5 indicates the recovery of matrix components, particularly in P5S5C. 10 The recovery was most significant in the P5S5C group; Masson staining results showed that collagen fibers in the model group were broken and disorganized, while those in the P5S5C group showed the most significant recovery. 10 The collagen fibers tend to be clearer and more densely and continuously arranged, indicating that the fiber ring repair scaffold of Example 1 is beneficial to improving the reconstruction effect of the fiber ring layer.

[0047] Further immunofluorescence was used to detect the expression of COL I in different regions, and the results are as follows: Figure 28 As shown; the expression of Aggrecan was detected by immunofluorescence, and the results are as follows. Figure 29 As shown; the expression of IL-1β was detected by immunohistochemistry, and the results are as follows. Figure 30 As shown, the effects of the scaffold on annulus fibrosus repair, extracellular matrix maintenance, and local inflammatory response were evaluated. COL I immunofluorescence results showed that COL I was continuously distributed in the annulus fibrosus region of the sham-operated group, while COL I expression was significantly reduced in the model group. 10 Both the P5S5 group and the P5S5 group were able to restore COL I expression, and the P5S5C group was able to restore COL I expression. 10 The recovery effect was better in the model group; Aggrecan immunofluorescence results showed that there was significant loss of proteoglycans in the model group, while P5S5C showed better recovery. 10The proteoglycans in the P5S5 group and the P5S5 group showed significant recovery, indicating that P5S5C 10 The loss of nucleus pulposus matrix was improved in both the P5S5 group and the P5S5 group, with P5S5C showing improved results. 10 The model group showed the best improvement in reducing nucleus pulposus matrix loss; immunohistochemical results of IL-1β showed that IL-1β was consistently highly expressed at 2 and 8 weeks in the model group, while P5S5C... 10 The significantly reduced IL-1β positivity rate in the group indicates that the annulus fibrosus repair scaffold of Example 1 also exhibits significant local anti-inflammatory effects in vivo. In summary, the annulus fibrosus repair scaffold of Example 1 is beneficial in promoting the restoration of the annulus fibrosus lamellar structure, maintaining extracellular matrix levels, and reducing local inflammation levels in vivo.

[0048] This specific embodiment is merely an explanation of this application and is not intended to limit it. After reading this specification, those skilled in the art can make modifications to this embodiment without contributing any inventive step, but such modifications are protected by patent law as long as they fall within the scope of the claims of this application.

Claims

1. A fiber ring repair scaffold, characterized in that: The nanofiber membrane comprises at least two layers, wherein the nanofibers in the nanofiber membrane are oriented in a specific direction and the orientation directions of the nanofibers in adjacent nanofiber membrane layers are intersected. The nanofiber membrane is obtained by cross-linking after directional electrospinning of a composite spinning solution. The composite spinning solution comprises silk fibroin, polyvinyl alcohol, chondroitin sulfate and a solvent. The weight ratio of the silk fibroin to the polyvinyl alcohol is 1:0.4-2.

5.

2. The annulus fibrosus repair scaffold according to claim 1, characterized in that: The weight ratio of the silk fibroin to the chondroitin sulfate is 1:0.05-0.

2.

3. The annulus fibrosus repair scaffold according to claim 1, characterized in that: The weight ratio of the silk fibroin to the solvent is 0.3-0.7:

1.

4. The annulus fibrosus repair scaffold according to claim 1, characterized in that: The average diameter of the nanofibers in the nanofiber membrane is 400-700 nm.

5. The annulus fibrosus repair scaffold according to claim 1, characterized in that: The preparation of the silk fibroin includes the following steps: taking silkworm cocoons, heating and degumming them under alkaline conditions, dissolving them in a lithium bromide solution to obtain a mixed solution, dialyzing the mixed solution to remove lithium bromide to obtain a silk fibroin solution, and freeze-drying the silk fibroin solution to obtain silk fibroin.

6. The annulus fibrosus repair scaffold according to claim 1, characterized in that: The included angle between the orientation directions of the nanofibers in the two adjacent nanofiber membranes is 20°-40°.

7. The annulus fibrosus repair scaffold according to claim 1, characterized in that: The directional electrospinning involves controlling the spinneret direction to be perpendicular to the radial direction of the roller collector, maintaining a constant rotation speed of the roller collector, and achieving directional alignment of nanofibers.

8. The annulus fibrosus repair scaffold according to claim 7, characterized in that: The parameters for the directional electrospinning are: voltage 15-17kV, spinneret liquid output speed 0.4-0.6mL / h, receiving distance 17-19cm, and rotation speed 1200-1400r / min.

9. A method for preparing a fibrous annulus repair scaffold as described in any one of claims 1-8, characterized in that: Includes the following steps: Polyvinyl alcohol and silk fibroin are dissolved in solvents to obtain polyvinyl alcohol solution and silk fibroin solution, respectively. Polyvinyl alcohol solution, silk fibroin solution and chondroitin sulfate are mixed and stirred evenly to obtain composite spinning solution. The composite spinning solution is subjected to directional electrospinning to obtain electrospun membrane. The electrospun membrane is cross-linked to obtain single-layer nanofiber membrane. Multiple nanofiber membranes are stacked in a cross-shaped manner with their fiber orientation directions to obtain the fiber ring repair scaffold.

10. The method for preparing a fiber annulus repair scaffold according to claim 9, characterized in that: The crosslinking treatment includes treatment with ethanol for 5-30 minutes and treatment with glutaraldehyde for 1-12 hours.

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