An annulus fibrosus repair material and stent for intervertebral discs and uses thereof

Abstract

This invention discloses an annulus fibrosus repair material and scaffold for intervertebral discs and their applications. The annulus fibrosus repair material comprises magnesium silicate nanofibers, tannic acid (TA), and transforming growth factor β (TGF-β). The magnesium silicate nanofibers contain self-assembled magnesium silicate nanosheets; the magnesium silicate nanosheets are encapsulated by tannic acid; and TGF-β is reversibly linked to the tannic acid via chemical bonds. This invention enhances extracellular matrix (ECM) expression in annulus fibrosus stem cells (AFSCs) by modulating the immune microenvironment.

Description

Technical Field

[0001] This invention belongs to the field of treatment technology for intervertebral disc degenerative diseases, specifically relating to an intervertebral disc annulus fibrosus repair material and scaffold, and their applications. Background Technology

[0002] The statements herein provide only background information in relation to this invention and do not necessarily constitute prior art.

[0003] In the treatment of degenerative disc diseases, minimally invasive discectomy is currently the standard clinical procedure for treating disc herniation and relieving nerve compression. However, this technique has a fundamental limitation: it can only remove the herniated diseased tissue to relieve symptoms, but it cannot repair or regenerate the annulus fibrosus that has been cut or damaged during surgery. This results in a non-healing "window" left on the annulus fibrosus after surgery, significantly increasing the risk of re-protrusion of the disc contents, with a recurrence rate as high as 5%-15%. In addition, the surgical trauma itself can trigger a local inflammatory response, further exacerbating the degenerative changes of the disc.

[0004] To address this challenge, several strategies have emerged in existing technologies to promote the repair of the annulus fibrosus, but all have significant limitations. For example, simple physical barriers or suture materials, such as fibrin glue and polyvinyl alcohol hydrogel, primarily function as mechanical sealants, lacking biological activity and unable to actively regulate the local pathological microenvironment (e.g., persistent inflammation). Furthermore, their degradation rate does not match the tissue regeneration rate, making true biological healing difficult and resulting in poor long-term efficacy. Traditional drug delivery systems: Some studies have attempted to load anti-inflammatory drugs or growth factors (such as TGF-β3) into hydrogels or microspheres and implant them into the defect. However, most of these systems lack intelligent responsiveness; drug release is not regulated by the local microenvironment, easily leading to burst release or insufficient release, and the short drug half-life makes it difficult to maintain a long-term effective therapeutic concentration at the lesion site.

[0005] Current technologies generally overlook the crucial initiation role of the immune response in tissue repair. The inflammatory microenvironment following annulus fibrosus injury (rich in pro-inflammatory M1 macrophages) is a core factor hindering stem cell function, leading to extracellular matrix metabolic imbalance, and tissue regeneration failure. Currently, there is a lack of effective means to actively and precisely regulate immune cell behavior, thereby transforming the destructive inflammatory environment into a regenerative and restorative one.

[0006] Most existing technologies focus only on one of the aforementioned single strategies (either blocking or drug delivery), failing to organically combine the two key aspects of "immune microenvironment regulation" and "intelligent drug delivery." This single-dimensional intervention is insufficient to break the vicious cycle of intervertebral disc degeneration and cannot create ideal biological conditions for the functional regeneration of the annulus fibrosus.

[0007] In summary, existing technologies for repairing annulus fibrosus defects mainly suffer from core defects such as limited biological function, uncontrollable drug release, neglect of immune regulation, and lack of multifunctional synergy. Summary of the Invention

[0008] In view of the shortcomings of the existing technology, the purpose of this invention is to provide a material and scaffold for repairing the annulus fibrosus of the intervertebral disc and its application, so as to fundamentally solve the problems that the existing technology has failed to overcome.

[0009] To achieve the above objectives, the present invention provides the following technical solution:

[0010] In a first aspect, the present invention provides an intervertebral disc annulus fibrosus repair material comprising magnesium silicon nanofibers, tannic acid (TA) and transforming growth factor β (TGF-β), wherein the magnesium silicon nanofibers comprise self-assembled magnesium silicon oxide nanosheets; the magnesium silicon oxide nanosheets are encapsulated by tannic acid; and TGF-β is reversibly bonded to the tannic acid.

[0011] Furthermore, tannic acid binds to magnesium in magnesium-silicon fibers through polyphenol-metal coordination.

[0012] In some embodiments of the present invention, the transforming growth factor β is selected from TGF-β1, TGF-β2 and TGF-β3.

[0013] In some embodiments of the present invention, the transforming growth factor β is TGF-β3; existing studies have shown that TGF-β3 mRNA is expressed in lymphocytes such as CD4+ T cells, CD8+ T cells, γδ T cells, and B cells. TGF-β3 participates in cell differentiation, embryogenesis, and development, and is crucial in tissue regeneration and scarless tissue repair.

[0014] Furthermore, TGF-β3 is linked to tannic acid by forming covalent bonds (e.g., Michell addition and Schiff base reaction).

[0015] In a second aspect, the present invention provides an intervertebral disc annulus fibrosus repair stent, comprising the intervertebral disc annulus fibrosus repair material described in the first aspect, wherein the magnesium-silicon nanofibers are prepared by the following method:

[0016] Step 1, Preparation of silica (SiO2) fiber membrane

[0017] S101, Preparation of spinning solution, by preparing a spinning solution from silicate or silanol sol;

[0018] In some embodiments of the present invention, the silicate is sodium silicate water glass, potassium silicate water glass, or sodium silicate nonahydrate; the silanol sol is a hydrolyzed sol of tetraethyl orthosilicate, methyltriethoxysilane, methyltrimethoxysilane, or phenyltrimethoxysilane in dilute acid.

[0019] S102, the spinning solution is mixed with an acidic solution, heated and stirred, and the resulting mixed solution is loaded into a spinning needle for wet spinning to prepare SiO2 fiber membrane.

[0020] In step S102, the rotational speed of the rotating receiving shaft is adjusted to obtain a randomly oriented SiO2 fiber film or a parallel-oriented SiO2 fiber film.

[0021] In step S102, the rotational speed of the rotating receiving shaft is adjusted to obtain a randomly oriented SiO2 fiber film at a lower speed of 50-150 r / min, and a parallel-oriented SiO2 fiber film at a higher speed of 1000-2000 r / min.

[0022] In a typical embodiment of the present invention, in step S102, the rotational speed of the rotating receiving shaft is adjusted to obtain a parallel-oriented SiO2 fiber film at a relatively high rotational speed of 1000-2000 r / min.

[0023] Step 2, Preparation of MgSi

[0024] Magnesium salt solution was mixed with ammonia water and transferred together with the SiO2 fiber membrane obtained in step 1 into a stainless steel high-pressure reactor lined with polytetrafluoroethylene. The mixture was then subjected to hydrothermal reaction at 130~150℃ for 8-12 hours to obtain MgSi.

[0025] In step 2, the magnesium salt solution is selected from magnesium chloride solution and magnesium nitrate solution.

[0026] In step 2, the mass ratio of the SiO2 fiber membrane to magnesium nitrate is 0.1-0.3g: 0.5-3g.

[0027] Thirdly, the present invention provides a method for preparing an intervertebral disc annulus fibrosus repair scaffold, comprising the following steps:

[0028] Step 3, Preparation of MgSi@TA

[0029] The MgSi product obtained in step 2 was immersed in Tris hydrochloride buffer, tannic acid was added, and the reaction was carried out to obtain MgSi@TA.

[0030] In step 3, the mass ratio of MgSi to tannic acid is 1:1~3.

[0031] In step 3, the Tris hydrochloride buffer has a molar concentration of 5–15 mM and a pH of 8.0–9.0. The reaction mixture is gently shaken at room temperature for 22–26 hours.

[0032] Step 4, Preparation of MgSi@TAT

[0033] The MgSi@TA obtained in step 3 is immersed in a solution containing transforming growth factor β (TGF-β), and the mixture is shaken to load the drug, thus obtaining MgSi@TAT, which is the intervertebral disc annulus fibrosus repair material.

[0034] In step 4, the mass ratio of MgSi@TA to transforming growth factor β is 1000:0.5~5.

[0035] Fourthly, the present invention provides the application of the intervertebral disc annulus fibrosus repair material described in the first aspect and the intervertebral disc annulus fibrosus repair material scaffold described in the second aspect in the regeneration of annulus fibrosus tissue.

[0036] In some embodiments of the present invention, experiments have shown that the intervertebral disc annulus fibrosus repair material scaffold promotes macrophage polarization toward M2; the present invention enhances extracellular matrix (ECM) expression of annulus fibrosus stem cells (AFSCs) by regulating the immune microenvironment.

[0037] The beneficial effects achieved by one or more embodiments of the present invention described above are as follows:

[0038] 1. This invention provides a material for repairing the annulus fibrosus of the intervertebral disc and provides an active immune regulation strategy: by designing a parallel-arranged nanofiber topology, it actively guides macrophages in vivo to polarize towards the anti-inflammatory M2 phenotype, fundamentally reversing the inflammatory microenvironment of intervertebral disc degeneration and breaking the vicious cycle of "inflammation-matrix degradation-re-damage".

[0039] 2. The intervertebral disc annulus fibrosus repair material provided by this invention enables intelligent drug delivery to the lesion microenvironment: utilizing the layered structure of magnesium silicate, a drug controlled-release system is constructed that responds to the acidic environment of the lesion area. This system can precisely release magnesium ions (Mg²⁺) at the site of inflammation. + It contains growth factors that promote the synthesis of extracellular matrix in fibroblasts (such as TGF-β3), and remains stable under normal physiological conditions, thereby improving efficacy and reducing side effects.

[0040] 3. The annulus fibrosus repair material provided by this invention can create a cascade effect of synergistic treatment: integrating the above-mentioned two major functions of "topological structure immune regulation" and "microenvironment-activated drug delivery". That is, firstly, a favorable microenvironment for tissue regeneration is created through immune regulation, and then bioactive factors are precisely released in this environment to jointly promote the vitality of annulus fibrosus stem cells (AFSCs), reverse their aging state, and ultimately efficiently regenerate annulus fibrosus tissue with normal biological function. Attached Figure Description

[0041] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0042] Figure 1 This describes the preparation process and characterization of an intervertebral disc annulus fibrosus repair material provided in Example 2; where a is the preparation method flow chart, b is a SEM image, c is a transmission electron microscope image, d is an XRD pattern, e is a full-scan XPS spectrum, f is a high-resolution N1s spectrum of MgSi@TAT, g is a high-resolution C1s spectrum of MgSi@TAT, h is an FT-IR spectrum, i is the TGF-β3 concentration in the supernatant during patch release, j is the cumulative TGF-β3 release over 14 days, and k is the Mg... 2+ Concentration, l is Mg 2+ Cumulative release.

[0043] Figure 2 This section describes the effect of patch topology on macrophage polarization in Example 3. Specifically, a represents a summary of the effects of different patch arrangements on macrophage polarization; b represents the effect of different patch arrangements on the relative mRNA expression level of INOS; c represents the effect of different patch arrangements on the relative mRNA expression level of IL-1β; d represents the effect of different patch arrangements on the relative mRNA expression level of CCR-7; e represents the effect of different patch arrangements on the relative mRNA expression level of CD206; f represents the effect of different patch arrangements on the relative mRNA expression level of ARG-1; g represents the effect of different patch arrangements on the relative mRNA expression level of IL-10; h represents the flow cytometry expression of different patch arrangements; i represents the immunofluorescence map of different patch arrangements; and j represents the statistical analysis of the immunofluorescence of different patch arrangements.

[0044] Figure 3This section illustrates the immunomodulatory effects of different patches on rat AFSCs and macrophages in a co-culture system, as described in Example 4. a is a schematic diagram of a Transwell culture system containing patches, AFSCs, and macrophages, with macrophages on the upper layer and AFSCs on the lower layer; bd shows the expression of TNF-α, IL-6, and IL-10 in the cell supernatant of different patch co-culture systems detected by enzyme-linked immunosorbent assay (ELISA); ef shows the mRNA expression of CDKN1A and CDKN2A; g shows β-galactosidase staining of different groups of AFSCs, with a scale bar of 250 μm; h shows the mRNA expression of COL-1α1, COL-2α1, Aggrecan, and MMP-13 in AFSCs detected in co-culture systems containing different patches; i shows immunofluorescence images of COL-1α1 and COL-2α1 in rat AFSCs, with a scale bar of 100 μm.

[0045] Figure 4 This is a study on the therapeutic effect of the rat-tail annulus fibrosus defect model in Example 5; where a is the process of constructing the rat-tail annulus fibrosus defect model and studying the therapeutic effect; b is CT examination; c is MRI examination; d is the statistical results of the relative intervertebral disc height (DHI) CT data at 4 weeks and 8 weeks; e is a schematic diagram of the compression test; f is the stress-strain curve of the compression test; g is the HE staining image of the sham surgery group; h is the HE staining image of the defect group; and i is the HE staining image of the P-MgSi@TAT treatment group.

[0046] Figure 5 The diameter distributions of R-MgSi and P-MgSi are shown.

[0047] Figure 6 This is a diagram of the EDS composition in Example 2. Detailed Implementation

[0048] It should be noted that the following detailed description is illustrative and intended to provide further explanation of the invention. Unless otherwise specified, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0049] As described in the background section, existing technologies generally overlook the crucial initiation role of the immune response in tissue repair. The inflammatory microenvironment following annulus fibrosus injury (rich in pro-inflammatory M1 macrophages) is a core factor hindering stem cell function, leading to extracellular matrix metabolic imbalance, and tissue regeneration failure. Currently, there is a lack of effective means to actively and precisely regulate immune cell behavior, thereby transforming the destructive inflammatory environment into a regenerative and restorative one.

[0050] This invention fundamentally reverses the inflammatory microenvironment of intervertebral disc degeneration by actively guiding macrophages in vivo to polarize towards the anti-inflammatory M2 phenotype, thus breaking the vicious cycle of "inflammation-matrix degradation-re-injury".

[0051] The present invention will be further described below with reference to the embodiments.

[0052] The reagents used in the examples were sourced from:

[0053] Tetraethyl orthosilicate (TEOS, 98.0%, Sinopharm Group, China).

[0054] Nitric acid (HNO3, 1N, Sinopharm Group, China).

[0055] Magnesium nitrate hexahydrate (Mg(NO3)2·6H2O, ≥ 99.0%, Sinopharm Group, China).

[0056] Ammonia water (NH3·H2O, 25-28%, Sinopharm Group, China).

[0057] Tannic acid (TA, 98%, Maclean's, China).

[0058] TGF-β3 (TGF-β3, Solarbio, China)

[0059] Example 1: Preparation of a material for repairing the annulus fibrosus of an intervertebral disc

[0060] Its preparation process is as follows Figure 1 As shown in Figure a, the steps include:

[0061] 1. Preparation of silica (SiO2) fiber membranes

[0062] First, 30 ml of tetraethyl orthosilicate was added to 30 ml of ethanol and mixed vigorously. Then, 4.5 ml of deionized water was added to the solution under magnetic stirring, and the mixture was stirred continuously for 0.5 hours. Next, 200 μL of nitric acid was slowly added dropwise. The temperature was raised to 60°C, and stirring was continued for 3 hours. At this point, the mixture was loaded into a plastic syringe equipped with a metal needle (size 21) for wet spinning to produce SiO2 fiber membranes. A high-voltage electric field (17 kV) was applied between the metal needle and the rotating receiving shaft, with a receiving distance of 15 cm and a constant feed rate of 4 mL / h. The rotational speed of the receiving shaft was adjusted; a lower speed of 100 r / min yielded randomly oriented SiO2 fiber membranes, while a higher speed of 1000 r / min yielded parallel-oriented SiO2 fiber membranes. Both types of SiO2 fiber membranes were transferred to a 60°C oven and kept for 12 hours to evaporate residual solvent. Finally, the fiber membrane was heated to 800°C at a heating rate of 1 °C / min and annealed in air for 5 hours.

[0063] 2. Preparation of MgSi

[0064] 3 g of magnesium nitrate hexahydrate (Mg(NO3)2·6H2O, ≥ 99.0%, Sinopharm Group, China) was dissolved in 80 ml of deionized water. Then, 2 ml of ammonia water was added to the solution. Finally, the resulting solution, along with the SiO2 fiber membrane, was transferred to a 100 mL stainless steel high-pressure reactor lined with polytetrafluoroethylene (PTFE). The reactor was hydrothermally reacted at 140°C for 10 hours to obtain the MgSi product. After the reactor cooled, the obtained MgSi product was washed three times with deionized water and ethanol, and then dried overnight at room temperature.

[0065] The parallel-oriented MgSi product was named P-MgSi, and the randomly oriented MgSi product was named R-MgSi.

[0066] 3. Preparation of MgSi@TA

[0067] To prepare MgSi@TA, 100 mg of the obtained MgSi product was immersed in 40 mL of 10 mM Tris hydrochloride buffer (pH=8.5), followed by the addition of 200 mg of tannic acid. The reaction mixture was gently shaken at room temperature for 24 hours. MgSi@TA prepared using P-MgSi was named P-MgSi@TA, and MgSi@TA prepared using R-MgSi was named R-MgSi@TA. Subsequently, MgSi@TA was separated, washed three times with deionized water, and dried overnight at room temperature for subsequent use.

[0068] 4. Preparation of MgSi@TAT

[0069] MgSi@TA (50 mg) was immersed in 50 mL of PBS solution containing 1 μg / mL recombinant human / mouse / rat TGF-β3 (TGF-β3, Solarbio, China). The mixture was gently shaken at 4°C for 12 hours to load the drug. MgSi@TAT prepared using P-MgSi@TA was named P-MgSi@TAT, and MgSi@TAT prepared using R-MgSi@TA was named R-MgSi@TAT. After drug loading, MgSi@TAT was separated and washed three times with PBS for subsequent testing. Following the same procedure, TGF-β3-loaded MgSi was also prepared and named MgSi@T.

[0070] Example 2: Characterization of a material for repairing the annulus fibrosus of an intervertebral disc

[0071] The morphology of magnesium silicon fibers (MgSi and MgSi@TAT) was imaged using scanning electron microscopy. Figure 1(b) P-type magnesium silicon exhibits a highly oriented fiber arrangement, while R-type magnesium silicon exhibits anisotropic fiber arrangement. Both types of flakes have similar fiber diameters, approximately 780 nanometers (nm). Figure 5 Magnesium-silicon fibers consist of numerous self-assembled magnesium-silicon-oxygen nanosheets on their surface. The exposed magnesium-silicon-oxygen nanosheets generate more active sites, significantly increasing their interaction with cells. TA can bind to magnesium in the magnesium-silicon fibers via polyphenol-metal coordination. After binding, the magnesium-silicon-oxygen nanosheets are encapsulated by a layer of TA, and the pores between the nanosheets are filled. Then, TGF-β3 is linked to TA through covalent bonding (e.g., Michell addition and Schiff base reactions). Transmission electron microscopy reveals a hollow tubular structure that facilitates degradation. Figure 1 (c) This can be attributed to Ostwald maturation or Kilkendr diffusion effects. These principles are commonly used to reconstruct the hollow structure of Sobel silica. After TA coating, an amorphous polymer layer appears on the MgSi@TA surface. Subsequent structural characterization was performed using p-type products. EDS composition mapping showed that the distribution of constituent elements in MgSi@TA was well uniform ( Figure 6 XRD pattern of MgSi ( Figure 1 (d) indicates that MgSiO3 nanosheets correspond to Mg3Si4O9(OH)4 (JCPDS No. 03-0174), while SiO2 fibers only exhibit the typical broad halo structure of amorphous SiO2. The surface elemental composition of MgSi, MgSi@TA, and MgSi@TAT was determined by XPS analysis. XPS was used to verify the multiple bonding structure of MgSi@TAT. The surface elemental composition of MgSi, MgSi@TA, and MgSi@TAT was determined by XPS analysis. XPS was used to verify the multiple bonding of MgSi@TAT. Full-scan XPS spectroscopy (...) Figure 1 In (e), only peaks of Mg, Si, and O were observed in MgSi, while a C1s peak belonging to TA appeared in MgSi@TA. Furthermore, an N1s peak belonging to TGF-β3 appeared in MgSi@TAT. More importantly, the high-resolution N1s spectrum was fitted and analyzed. Figure 1 (f). For MgSi@TAT, the peaks at 399.64 eV, 400.24 eV, and 401.54 eV are attributed to CN, C=N, and C-NH3, respectively. + The presence of C–N and C=N bonds confirms the Michael addition reaction and Schiff base formation between TA and TGF-β3. Furthermore, the high-resolution C1s spectrum of MgSi@TAT ( Figure 1 The MgSi@TA sample (g) exhibits new CN-related peaks compared to MgSi@TA, indicating that TGF-β3 has been grafted onto MgSi@TA. FT-IR spectra ( Figure 1 The result is also proven by h). 1022cm -1 The peak at 1723 cm⁻¹ represents the Si-O-Si stretching vibration of MgSi. Compared to MgSi, MgSi@TA exhibits a peak at 1723 cm⁻¹. -1 A peak appeared at 1650 cm⁻¹, corresponding to the stretching vibration of C=O in TA, indicating that MgSi is coated with TA. Furthermore, at 1650 cm⁻¹... -1 and 1513cm -1 The peaks at the specified locations belong to the C=N group in the Schiff group and the CN group in the Michael addition reaction, respectively, both originating from MgSi@TAT. In summary, the quinone groups in the TA coating layer can serve as active sites, undergoing cross-linking reactions with the amino group (-NH2) of TGF-β3 via Michael addition / Schiff group. The loading of TA and TGF-β3 was demonstrated by measuring the Zeta potential. The Zeta potential of MgSi@TA decreased to -21.37 mV, indicating that TA was effectively loaded onto MgSi, due to the abundance of phenolic hydroxyl and quinone groups in TA. The loading of TGF-β3 further reduced the potential to -25.83 mV, which can be attributed to the carboxyl group (-COOH) of TGF-β3. To evaluate the role of TA in this system, the drug delivery performance of MgSi and MgSi@TA was compared. The MgSi patch loaded only with TGF-β3 without using TA as a medium was named MgSi@T. During release, the real-time monitored concentration of MgSi@TAT was consistently higher than that of MgSi@T (the MgSi patch loaded with TGF-β3). After loading TGF-β3, the residual concentrations of the supernatant in the MgSi@T group and the MgSi@TAT group were 6.34 and 14.82 ng / mL, respectively. Figure 1 (i). It can be concluded that MgSi@T adsorbs more TGF-β3 than MgSi@TAT. In the MgSi@TAT group, the real-time concentration of TGF-β3 remained within the therapeutic range throughout the measurement period. After 14 days of release, the cumulative release of MgSi@TA was also significantly higher than that of MgSi@T. The MgSi@TAT group was still able to release more TGF-β3, although its loading was lower than that of the MgSi@T group (i). Figure 1(j). Compared to MgSi@T, the high release rate of MgSi@TAT may be attributed to the reversible chemical bond linkage of TA-TGF-β3. This reversible chemical bond endows the system with the ability to release a concentration gradient. Therefore, in the simulated release process, drug uptake leads to the breaking of the TA-TGF-β3 bond and sustained drug release. With the development of intervertebral disc herniation (IVDH), the microenvironment exhibits characteristics such as decreased glucose, oxygen, and pH, which play a crucial role in cell survival and matrix renewal. Therefore, designing biomedical materials for the treatment of IVDH based on these properties is a very promising approach. Figure 1 As shown in k and l in 1, the released Mg 2+ The real-time concentration ranged from 0.5 to 2.5 mM. This range is within the safe range and close to physiological levels. Mg 2+ The release rate of Mg increases with increasing acidity. At pH 7.4, Mg 2+ The release rate was much lower at pH 6.0 and 5.0, while the release curves at pH 6.0 and 5.0 were quite similar. With the increase of Mg... 2+ Increased release due to Mg 2+ Through coordination with TA, more TA-TGF-β3 also detaches from the MgSi fibers. This indicates that the system exhibits good pH-responsive release performance of TA and TGF-β3. Furthermore, Schiff bases also exhibit acid instability. Within the IVDH microenvironment, the drug bound to it can be easily released from the patch through bond breaking.

[0072] Example 3: The effect of patch topology on macrophage polarization

[0073] The topological structure of bio-scaffold materials has a significant impact on macrophage polarization. This study investigated the effects of patches with different arrangement patterns (random and parallel) on macrophage polarization. Figure 2 (a) Macrophages were cultured on fibrous scaffolds with different arrangements for 2 days, and then RNA was extracted. Six representative macrophage polarization markers were analyzed by PCR: ARG-1, CD206, IL-10, IL-1β, iNOS, and CCR-7. Results ( Figure 2The results (bg) showed that R-MgSi promoted the expression of IL-1β, iNOS, and CCR-7, while inhibiting the expression of ARG-1, CD206, and IL-10, indicating macrophage polarization towards the M1 phenotype. In contrast to R-MgSi, the expression of these markers was reversed in P-MgSi, thereby promoting macrophage polarization towards M2. This suggests that fibrillary arrangement can effectively influence macrophage polarization, and the method exhibits stability and safety. Biophysical signals, including morphological and topological structures, can be transduced into indirect signals via integrin-mediated pathways. These interactions convert the physicochemical and mechanical signals of surface nanoscale features into biological signals, thereby regulating the local biological microenvironment and cellular responses. Furthermore, R-MgSi@TAT also promoted macrophage polarization towards M2 to some extent, exhibiting a similar pattern to P-MgSi. This may be attributed to the anti-inflammatory and antioxidant properties of TA and TGF-β3. Notably, P-MgSi@TAT exhibited the highest expression levels of these M2 markers and the lowest expression levels of M1 markers, indicating its optimal ability to promote macrophage polarization towards M2.

[0074] By flow cytometry ( Figure 2 (h) and immunofluorescence ( Figure 2 (i) Macrophage polarization was further evaluated by calculating the CD206+ / CD86+ ratio to assess macrophage polarization. The results showed that the order of ability to promote M2 polarization was: P-MgSi@TAT > P-MgSi > R-MgSi@TAT > R-MgSi (i) Figure 2 (j). These findings are consistent with PCR data, indicating that P-MgSi@TAT can better promote M2 macrophage polarization. Typically, cells utilize integrin-ligand interactions to probe, attach to, and respond to the topology of a matrix, actively reshaping their cytoskeleton network and forming focal adhesions, thereby influencing cell shape, motility, and function. Studies have found that topology typically regulates macrophage expression and polarization through the YAP / TAZ, MRTF-A, PI3K / AKT, JAK-STAT, and NF-κB signaling pathways. Since some of these pathways are highly expressed in macrophages, topology may have a more significant regulatory effect on macrophages compared to other cells. Therefore, manipulating the surface properties of patches by regulating their topology and morphology to modulate the immune microenvironment and thereby enhance ECM expression in AFSCs is a promising and meaningful approach for future research.

[0075] Example 4: Effect of patch on extracellular matrix metabolism of AFSCs

[0076] The effects of patches with different structures and compositions on the ECM expression of AFSCs were further investigated. Figure 3(e). Rat macrophages were seeded in Transwell chambers, and different treated patches were placed on the surface of the chambers. AFCs were then seeded in six-well plates. The upper Transwell chambers were then placed on the top layer of the six-well plates and cultured for 2-3 days before subsequent experiments.

[0077] PCR analysis showed that, compared with R / P-MgSi, the expression of COL-1α1, COL-2α1, and Aggrecan was increased in R / P-MgSi@TAT after loading with TA and TGF-β3, while the expression of MMP-13 was decreased. Figure 3 These findings indicate that the gradual release of TGF-β3 from the patch effectively promotes ECM synthesis while inhibiting its degradation. However, no significant differences were observed between R-MgSi and P-MgSi, or between R-MgSi@TAT and P-MgSi@TAT, suggesting that fiber arrangement structure has no significant effect on ECM expression in AFSCs. Western blot analysis yielded similar results to PCR. After loading with TA and TGF-β3, the expression levels of COL-1α1, COL-2α1, and Aggrecan increased, while the expression of MMP-13 decreased ( Figure 3 (fj). Similarly, consistent with PCR results, fiber arrangement (parallel and random) did not appear to affect the expression of these four proteins, as no significant differences were observed. IF staining (immunofluorescence staining) of COL-1α1 and COL-2α1, Figure 2 The results also showed that R / P-MgSi exhibited higher expression levels than the untreated group after loading with TA and TGF-β3. In summary, these results indicate that MgSi effectively promotes ECM synthesis and inhibits its degradation through the combined action of TA and TGF-β3. This dual mechanism, involving the regulation of the immune microenvironment and sustained release of TGF-β3, promotes normal ECM metabolism in AFSCs.

[0078] Example 5: Therapeutic effect on a rat-tail type annulus fibrosus defect model

[0079] Male SD rats (average weight 300-350 g) were used to study the repair and regeneration of the annulus fibrosus of the intervertebral disc in vivo. The rats were purchased from Vital River (China). All animal experiments were conducted in accordance with the National Institutes of Health's "Guidelines for the Care and Use of Laboratory Animals" and were approved by the Institutional Animal Care and Use Committee of Qilu Hospital, Shandong University (ethics approval number: DWLL-2023-140). The entire experimental process strictly followed the ARRIVE guidelines for animal research.

[0080] Construction of a rat model of annulus fibrosus defect: Male SD rats were anesthetized by intraperitoneal injection of an anesthetic. After thorough disinfection, a transverse incision was made in the center of the surgical area, and the soft tissue was carefully dissected to expose the intervertebral disc. Part of the annulus fibrosus was removed to create a rectangular defect measuring 2.0 mm long, 1.0 mm wide, and 1.0 mm deep to simulate an acute annulus fibrosus defect. The defect area was covered with eight layers of P-MgSi@TAT with an area similar to that of the defect area. The layered patch was fixed to the tissue using 3MVetbond tissue adhesive. 3MVetbond has been reported to not induce inflammation; it was applied to the four corners of the patch to aid in fixation. Penicillin was administered once daily for three consecutive days postoperatively.

[0081] This embodiment establishes a rat-tail type annulus fibrosus defect model ( Figure 4 (a) A portion was removed from the annulus fibrosus and repaired with this material (P-MgSi@TAT). CT and MRI scans were performed at 4 and 8 weeks to observe the height of the intervertebral disc ( Figure 4 (middle bd). The results showed that, compared with the sham-operated group, the intervertebral disc height in the defective group was significantly reduced, while the intervertebral disc height of mice treated with P-MgSi@TAT showed significant recovery. These imaging results indicate that P-MgSi@TAT has a significant ability to repair annular defects. To investigate the recovery of biological function after repairing the annulus fibrosus with P-MgSi@TAT, we performed compression tests on the vertebral segments containing the repaired intervertebral discs. Figure 4 (e). Previous studies have shown that compressibility characteristics are closely related to the functional integrity of the intervertebral disc, such as its ability to withstand bending and compression. Typical stress-strain curves of the sham-operated group ( Figure 4 As shown in (e), healthy intervertebral discs can buffer stress to a certain extent and dissipate stress through deformation within a specific range. In contrast, the stress in the defective group increased rapidly with increasing strain, while the experimental group showed intermediate results. Compression tests indicated that the compressive modulus of the toe and linear regions in the defective group was significantly increased, possibly due to the formation of scar tissue. However, the compressive modulus of the P-MgSi@TAT group was similar to that of the sham-operated group, indicating that P-MgSi@TAT effectively restored the biomechanical properties of the intervertebral disc.

[0082] These results indicate that P-MgSi@TAT can repair annulus fibrosus defects and maintain the biomechanical integrity of the intervertebral disc. To further evaluate the repair of the annulus fibrosus (AF), the intervertebral disc was removed from the surgical site 8 weeks postoperatively for histological analysis. HE staining was performed to examine the morphology of the AF, and IF was used to assess the expression of COL-1α1 and COL-2α1. Figure 4HE staining showed that the AF in the defective group had obvious defects and poor healing, while the healing in the P-MgSi@TAT group was improved compared with the defective group. IF analysis showed that the expression levels of COL-1α1 and COL-2α1 were low in the defective group, while the expression levels of these two collagens were significantly higher in the P-MgSi@TAT group (P<0.05). Histological staining showed that the implanted composite patch effectively promoted the synthesis of extracellular matrix, thereby promoting AF repair.

[0083] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. An annulus fibrosus repair material for intervertebral discs, characterized by, The invention comprises magnesium silicon nanofibers, tannic acid, and transforming growth factor β. The magnesium silicon nanofibers contain self-assembled magnesium silicon oxide nanosheets, which are encapsulated by tannic acid. The transforming growth factor β is TGF-β3, which is covalently linked to the tannic acid. The magnesium-silicon nanofibers were prepared by the following method: Step 1, Preparation of silica fiber membrane: S101, Preparation of spinning solution, by preparing a spinning solution from silicate or silanol sol; S102, the spinning solution is mixed with an acidic solution, heated and stirred, and the resulting mixed solution is loaded into a spinning needle for wet spinning to prepare SiO2 fiber membrane; Step 2, Preparation of MgSi: A magnesium salt solution was mixed with ammonia water and transferred together with the SiO2 fiber membrane obtained in step 1 into a stainless steel high-pressure reactor lined with polytetrafluoroethylene. The mixture was then subjected to a hydrothermal reaction at 130-150°C for 8-12 hours to obtain MgSi. The preparation method of the intervertebral disc annulus fibrosus repair material further includes the following steps: Step 3, Preparation of MgSi@TA: The MgSi product obtained in step 2 was immersed in Tris hydrochloride buffer, tannic acid was added, and the reaction was carried out to obtain MgSi@TA; Step 4, Preparation of MgSi@TAT: The MgSi@TA obtained in step 3 is immersed in a solution containing transforming growth factor β and subjected to shaking treatment to load the drug, thus obtaining MgSi@TAT, which is the intervertebral disc annulus fibrosus repair material.

2. The intervertebral disc annulus fibrosus repair material according to claim 1, characterized in that, Tannic acid binds to magnesium in magnesium-silicon fibers through polyphenol-metal coordination.

3. A scaffold for repairing the annulus fibrosus of an intervertebral disc, characterized in that, It includes the intervertebral disc annulus fibrosus repair material as described in any one of claims 1 to 2.

4. The intervertebral disc annulus fibrosus repair stent according to claim 3, characterized in that, In step S102, the rotational speed of the rotating receiving shaft is adjusted to obtain a randomly oriented SiO2 fiber film or a parallel-oriented SiO2 fiber film.

5. The intervertebral disc annulus fibrosus repair stent according to claim 3, characterized in that, In step S102, the rotational speed of the rotating receiving shaft is adjusted to obtain a parallel-oriented SiO2 fiber film at a speed of 1000-2000 r / min.

6. The application of the intervertebral disc annulus fibrosus repair scaffold according to claim 3 in annulus fibrosus tissue regeneration.

7. The application of the intervertebral disc annulus fibrosus repair scaffold according to claim 3 in annulus fibrosus tissue regeneration, characterized in that, The intervertebral disc annulus fibrosus repair scaffold promotes macrophage polarization towards M2; and enhances extracellular matrix expression of annulus fibrosus stem cells by regulating the immune microenvironment.

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