Composite immune scaffold for promoting repair of injured spinal cord and application of composite immune scaffold

Abstract

The invention relates to the field of medical instruments, and provides a composite immune scaffold for promoting spinal cord repair after injury and application, the composite immune scaffold is formed by printing through a three-dimensional printing technology, the composite immune scaffold comprises at least two matrix layers and at least one support layer, the support layer is located between the at least two matrix layers, and the matrix layers are arranged on the support layer. IRF4 proteins are distributed inside and / or on the surfaces of the supporting layer and the matrix layer. When the composite immune scaffold is delivered to a spinal cord injury part, the matrix layer and the supporting layer can fill the injury area to maintain the spinal cord contour structure, and meanwhile, the matrix layer and the supporting layer can well bear IRF4 protein, so that IRF4 is enriched at the spinal cord injury part, and the IRF4 protein can be continuously released at the spinal cord injury part; the compound can inhibit the polarization of M1 type macrophages in an immune microenvironment and promote the polarization of M2 type macrophages, is beneficial to the formation of a microenvironment for nerve regeneration, promotes the regeneration of neurons and axons, inhibits the formation of glial scars, and promotes the repair after spinal cord injury.

Description

Technical Field

[0001] This application relates to the field of medical device technology, and more specifically, to a composite immune scaffold for promoting spinal cord injury repair and its application. Background Technology

[0002] Spinal cord injury (SCI) is a central nervous system disorder characterized by loss of sensory and motor function below the level of spinal cord injury, urinary and fecal incontinence, and sexual dysfunction, placing a heavy burden on individuals, families, and society. Following SCI, a series of inflammatory responses occur, including: 1) activation of microglia; 2) neutrophil infiltration; 3) polarization of macrophages / microglia; 4) release of cytokines; and 5) oxidative stress.

[0003] Macrophage polarization is considered a major cause of microenvironmental imbalance and hinders neurogenesis. Macrophages typically exist in two phenotypes: M1 and M2. During the acute phase of spinal cord injury, M1 macrophages secrete pro-inflammatory cytokines, initiating an inflammatory cascade. Conversely, M2 macrophages are generally considered an anti-inflammatory subtype, capable of suppressing the inflammatory response.

[0004] Interferon regulatory factor 4 (IRF4) is a key regulator of macrophages, playing a crucial role in regulating the inflammatory response. As an important transcription factor in immunity, IRF4 competitively promotes M2 macrophage polarization and binding to MyD88, thereby addressing inflammation and tissue repair in spinal cord injury. IRF4 improves the inflammatory microenvironment and functional recovery by disrupting IRF5-MyD88 and ultimately promoting STAT6-mediated M2 polarization. However, in improving spinal cord injury, the residence time of IRF4 protein at the injury site is short, making it difficult to exert a sustained effect. Moreover, the effect of IRF4 in improving the inflammatory microenvironment and functional recovery is limited, and its repair function for spinal cord injury cannot meet the required level.

[0005] Therefore, further improving the duration and effectiveness of IRF4 is particularly important in the field of spinal cord injury. Summary of the Invention

[0006] In view of this, embodiments of this application provide a composite immune scaffold for promoting spinal cord injury repair and its application, which can mimic the structure and function of the spinal cord, ensure the continuous release of IRF4 protein at the site of spinal cord injury, inhibit the polarization of M1 macrophages in the immune microenvironment and promote the polarization of M2 macrophages, promote neuronal and axon regeneration, and inhibit glial scar formation.

[0007] In a first aspect, embodiments of this application provide a composite immune scaffold that promotes repair after spinal cord injury. The composite immune scaffold is formed by three-dimensional printing technology. The composite immune scaffold includes at least two matrix layers and at least one support layer. The support layer is located between the at least two matrix layers. IRF4 protein is distributed inside and / or on the surface of the support layer and the matrix layer.

[0008] In some embodiments, the concentration of the IRF4 protein is 100 ng / ml.

[0009] In some embodiments, the materials used to print the composite immune scaffold include gelatin hydrogel containing grafting functional groups, a blue light initiator, and the IRF4 protein.

[0010] In some embodiments, the support layer includes a plurality of support units arranged at intervals, with pores formed between the plurality of support units.

[0011] In some embodiments, the contact angle of the composite immune scaffold of this application is less than 90°.

[0012] Secondly, embodiments of this application provide an application of the composite immune scaffold described in the first aspect for promoting spinal cord injury repair, wherein the composite immune scaffold can inhibit M1 macrophage polarization and promote M2 macrophage polarization.

[0013] In some embodiments, the composite immune scaffold can promote the secretion of anti-inflammatory factor IL-4, inhibit the secretion of pro-inflammatory factor IL-6, and regulate the immune microenvironment after spinal cord injury.

[0014] In some embodiments, after the composite immune scaffold is implanted at the site of spinal cord injury, the IRF4 protein can be continuously released for 28 days.

[0015] In some embodiments, after the composite immune scaffold is implanted at the site of spinal cord injury, the composite immune scaffold can preserve the spatial structure of the spinal cord and promote the integration of the composite immune scaffold with the host tissue within the spinal cord.

[0016] In some embodiments, after the composite immune scaffold is implanted at the site of spinal cord injury, the composite immune scaffold can promote the effective recovery of motor, sensory, urinary functions and neurophysiological conduction after spinal cord injury.

[0017] In some embodiments, after the composite immune scaffold is implanted at the site of spinal cord injury, the composite immune scaffold can promote the regeneration of neurons and axons and reduce the formation of glial scars.

[0018] The technical solution of this application has at least the following beneficial effects:

[0019] The composite immune scaffold provided in this application promotes the repair of spinal cord injury. The composite immune scaffold includes a matrix layer and a support layer. The support layer supports at least two matrix layers. IRF4 protein is distributed inside and / or on the surface of the support layer and the matrix layer. When the composite immune scaffold is delivered to the spinal cord injury site, the matrix layer and the support layer can fill the injured area and maintain the spinal cord contour structure. At the same time, the matrix layer and the support layer can effectively carry IRF4 protein and enable IRF4 to accumulate at the spinal cord injury site. IRF4 protein can be continuously released at the spinal cord injury site, inhibiting the polarization of M1 macrophages and promoting the polarization of M2 macrophages in the immune microenvironment. This is conducive to the formation of a microenvironment for nerve regeneration, promoting neuronal and axonal regeneration, inhibiting glial scar formation, and promoting the repair of spinal cord injury. Attached Figure Description

[0020] Figure 1 A perspective view of the composite immune scaffold provided in the embodiments of this application;

[0021] Figure 2 This is a front view of the composite immune scaffold provided in an embodiment of this application;

[0022] Figure 3 Molecular structure diagrams of IRF4, IRF5, MyD88, and STAT6 provided for embodiments of this application;

[0023] Figure 4A Images of the composite immune scaffold provided in the embodiments of this application;

[0024] Figure 4B SEM image of the composite immune scaffold provided in the embodiments of this application;

[0025] Figure 4C Rheological analysis of the composite immune scaffold provided in the embodiments of this application Figure 1 ;

[0026] Figure 4D Rheological analysis of the composite immune scaffold provided in the embodiments of this application Figure 2 ;

[0027] Figure 4E This is a diagram showing the contact angle measurement results of the composite immune scaffold provided in the embodiments of this application;

[0028] Figure 4F Drug release curve of the composite immune scaffold provided in the embodiments of this application

[0029] Figure 5 The survival status of BM-MSCs on the composite immune scaffold at days 7, 14, and 28, as provided in the embodiments of this application;

[0030] Figure 6AThe diagram shows the potential results of rats after limb amputation and implantation in the Sham group, PBS group, 3D Scaffold group, IRF4 group and 3D Scaffold@IRF4 group, respectively, as provided in this embodiment.

[0031] Figure 6B The statistical results of the potentials of rats implanted after limb amputation in the Sham group, PBS group, 3D Scaffold group, IRF4 group and 3D Scaffold@IRF4 group provided in this embodiment are as follows:

[0032] Figure 6C The BBB scores of the rat limb amputation implantation groups (PBS group, 3D Scaffold group, IRF4 group, and 3DScaffold@IRF4 group) provided in this embodiment are shown.

[0033] Figure 6D The hind limb support angle was recorded weekly after surgery for the rat limb amputation implantation in the PBS group, 3D Scaffold group, IRF4 group and 3DScaffold@IRF4 group respectively.

[0034] Figure 6E The Von Frey scores of the left hind limbs after limb amputation in rats in this embodiment were obtained from the PBS group, 3D Scaffold group, IRF4 group and 3DScaffold@IRF4 group respectively.

[0035] Figure 6F The Von Frey scores of the right hind limb after limb amputation in rats in this embodiment were obtained from the PBS group, 3D Scaffold group, IRF4 group, and 3DScaffold@IRF4 group, respectively.

[0036] Figure 6G The urine output within 8 axes after limb amputation in rats implanted with PBS, 3D Scaffold, IRF4 and 3DScaffold@IRF4 were measured in this embodiment.

[0037] Figure 6H The results of blood urea nitrogen and serum creatinine levels in the PBS group, 3D Scaffold group, IRF4 group and 3DScaffold@IRF4 group after rat limb amputation provided in this example;

[0038] Figure 7A Quantitative analysis of macrophages in rats implanted in the PBS group, 3D Scaffold group, IRF4 group and 3D Scaffold@IRF4 group 8 weeks after implantation into the spinal cord injury site;

[0039] Figure 7B Quantitative analysis of anti-inflammatory macrophages in rats after 8 weeks of implantation into the spinal cord injury sites of the PBS group, 3D Scaffold group, IRF4 group and 3D Scaffold@IRF4 group provided in this embodiment;

[0040] Figure 7C The quantitative analysis results of M1 macrophages, M2 macrophages, cytokine IL-6 and cytokine IL-4 eight weeks after the PBS group, 3D Scaffold group, IRF4 group and 3D Scaffold@IRF4 group were implanted into the spinal cord injury site of rats.

[0041] Figure 8A H&E staining images of the PBS group, 3D Scaffold group, IRF4 group and 3D Scaffold@IRF4 group after implantation into the spinal cord injury site of rats, respectively, provided in this embodiment;

[0042] Figure 8B NeuN immunostaining images of spinal cord neurons implanted in the spinal cord injury sites of rats in the PBS group, 3D Scaffold group, IRF4 group and 3D Scaffold@IRF4 group provided in this embodiment;

[0043] Figure 8C GFAP immunostaining images of astrocytes in the spinal cord tissue after implantation into the spinal cord injury sites of rats in the PBS group, 3D Scaffold group, IRF4 group and 3D Scaffold@IRF4 group provided in this embodiment;

[0044] Figure 9 Representative immunofluorescence staining images of β-III tubulin and NeuN (neuron-specific enolase) after implantation into the spinal cord injury sites of rats in the PBS group, 3D Scaffold group, IRF4 group and 3D Scaffold@IRF4 group provided in this embodiment;

[0045] Figure 10A The molecular dynamics simulation results of the IRF4-MyD88 complex provided in this embodiment;

[0046] Figure 10B The molecular dynamics simulation results of the IRF5-MyD88 complex provided in this embodiment;

[0047] Figure 10C The molecular dynamics simulation results of the IRF4-MyD88-IRF5 complex provided in this embodiment;

[0048] Figure 10DThe molecular dynamics simulation results of the IRF4-STAT6 complex provided in this embodiment;

[0049] Figure 10E Western blot images of JAK1, STAT6, and IRF4 protein expression in different groups provided in this embodiment;

[0050] Figure 10F The quantitative analysis results of JAK1, STAT6 and IRF4 protein expression in different groups provided in this embodiment;

[0051] Figure 10G Western blots of IRF5 and STAT5 protein expression in different groups provided in this embodiment;

[0052] Figure 10H The quantitative analysis results of IRF5 and STAT5 protein expression in different groups provided in this embodiment;

[0053] Figure 10I A comparison of the relative expression levels of the components JAK1, STAT6, IRF4, IRF5 and STAT5 provided in this embodiment. Detailed Implementation

[0054] The following are preferred embodiments of the present invention. It should be noted that, for those skilled in the art, several improvements and modifications can be made without departing from the principle of the embodiments of the present invention, and these improvements and modifications are also considered to be within the protection scope of the embodiments of the present invention.

[0055] The concepts involved in this application are explained below.

[0056] IRF4 (Interferon Regulatory Factor 4) is a transcription factor belonging to the IRF family. It plays a crucial role in the immune system, primarily regulating the differentiation, proliferation, apoptosis, and immune responses of immune cells. IRF4 is mainly expressed in immune cells such as lymphocytes, dendritic cells, and macrophages. IRF4 is also known as...

[0057] MUM1, LSIRF, Pip, ICSAT, etc.

[0058] IRF5 (Interferon Regulatory Factor 5) is a transcription factor belonging to the interferon regulatory factor (IRF) family. IRF5 plays a crucial role in the immune system, primarily involved in regulating the activation, differentiation, and inflammatory responses of immune cells.

[0059] MyD88 (Myeloid Differentiation Primary Response Protein 88) is a key adaptor protein that plays an important role in signal transduction in the immune system, especially in the innate immune response. It is a core component of the Toll-like receptor (TLR) and interleukin-1 receptor (IL-1R) signaling pathways.

[0060] STAT6 (Signal Transducer and Activator of Transcription 6) is a cytoplasmic transcription factor belonging to the STAT family of signal transducers and activators of transcription. STAT6 plays a crucial role in cytokine signaling, particularly in regulating immune responses and cell differentiation. It is primarily involved in signaling pathways mediated by interleukin-4 (IL-4) and interleukin-13 (IL-13).

[0061] BM-BSCs are a type of bone marrow mesenchymal stem cells, a cell type commonly used in bioprinting.

[0062] In related technologies, IRF4 protein is introduced into cells at the site of spinal cord injury to increase its expression, particularly promoting macrophage polarization towards the anti-inflammatory phenotype (M2 type), thereby alleviating inflammation and promoting tissue repair. However, in other technologies, IRF4 protein is delivered to the spinal cord injury site via intravenous or intraperitoneal injection. Due to the lack of targeting specificity in these methods, IRF4 protein is distributed throughout the body via the bloodstream, making it difficult to specifically accumulate at the spinal cord injury site. Oral injection of IRF4 protein to the spinal cord injury site is also problematic. The presence of cerebrospinal fluid circulating around the spinal cord and its continuous flow and renewal rapidly dilutes and carries away the injected IRF4 protein, resulting in a short residence time at the spinal cord injury site and hindering its sustained effectiveness.

[0063] Based on this, embodiments of this application provide a composite immune scaffold that promotes spinal cord repair after injury. Figure 1 A three-dimensional view of the composite immune scaffold provided in the embodiments of this application, as shown below. Figure 1 As shown, the composite immune scaffold includes at least two matrix layers 1 and at least one support layer 2, with the support layer 2 disposed between the at least two matrix layers 1. IRF4 protein (IRF4 protein is distributed on the interior and / or surface of the support layer and the matrix layer) is present in the matrix layer. Figure 1 (Not shown in the image).

[0064] The composite immune scaffold provided in this application promotes the repair of spinal cord injury. The composite immune scaffold includes a matrix layer 1 and a support layer 2. The support layer 2 supports at least two matrix layers 1. IRF4 protein is distributed inside and / or on the surface of the support layer and the matrix layer. When the composite immune scaffold is delivered to the spinal cord injury site, the matrix layer 1 and the support layer 2 can fill the injured area and maintain the spinal cord contour structure. At the same time, the matrix layer 1 and the support layer 2 can effectively carry IRF4 protein and also enable IRF4 to accumulate at the spinal cord injury site. IRF4 protein can be continuously released at the spinal cord injury site, inhibiting the polarization of M1 macrophages and promoting the polarization of M2 macrophages in the immune microenvironment. This is conducive to the formation of a microenvironment for nerve regeneration, promoting nerve and axon regeneration, inhibiting glial scar formation, and promoting the repair of spinal cord injury.

[0065] The composite immune scaffold of this application is formed by 3D printing technology. Through research, it has been found that the composite immune scaffold formed by 3D printing technology has good mechanical and mechanical properties and can fill the damaged area to maintain the spinal cord contour structure.

[0066] In some implementations... Figure 2 This is a front view of the composite immune scaffold provided in an embodiment of this application, such as... Figure 2 As shown, the support layer 2 includes multiple support units 21 arranged at intervals, with pores 22 formed between the multiple support units 21. The pores 22 formed between the multiple support units 21 in this application can reserve a certain space for the material of the printed composite immune scaffold to absorb water and expand, thereby avoiding damage to the spinal cord injury site caused by the expansion of the composite immune scaffold by water absorption.

[0067] In some embodiments, the printed composite material includes a gelatin hydrogel containing grafting functional groups (MA), a blue light initiator, and an IRF4 protein. Specifically, the gelatin is methacrylic anhydride gelatin (GelMA), and the blue light initiator is lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP). During the printing process, the blue light initiator is used to initiate the polymerization of the gelatin hydrogel containing grafting functional groups (MA). The composite immune scaffold of this application has a simple and clear composition, excellent mechanical properties and biocompatibility, which is beneficial for reducing graft immune rejection.

[0068] A digital model of the spinal cord hemisection to be repaired can be obtained based on medical imaging data. A composite immune scaffold can then be printed using 3D printing technology. This scaffold has a structural size suitable for the axial regeneration and extension of neurons and can load an appropriate amount of IRF4 protein on its surface and inside. The composite immune scaffold is then implanted into the site to be repaired. The composite immune scaffold can support the spatial structure of the site to be repaired and can also promote the integration of the composite immune scaffold with the host cells of the site to be repaired. At the same time, the IRF4 protein can be continuously released at the site to be repaired, promoting neuronal regeneration and reducing the formation of glial scars.

[0069] In some embodiments, the contact angle of the composite immune scaffold of this application is less than 90°, meaning that the composite immune scaffold of this application has good hydrophilicity. The hydrophilic surface of the composite immune scaffold can mimic the polarity of the extracellular matrix, thereby enhancing cell adhesion, spreading, and proliferation. In addition, the hydrophilicity of the composite immune scaffold of this application reduces the resistance to cell contact, thereby accelerating tissue regeneration and repair processes, promoting the diffusion of water molecules, and helping to regulate the sustained release of IRF4 on the composite immune scaffold.

[0070] The specific preparation process of the composite immune scaffold of this application is described in detail below.

[0071] The preparation method of the composite in this application includes the following steps:

[0072] A mixture of 10% lyophilized GelMA hydrogel powder (GelMA, SP-BI-G01-2, SunP (Beijing) Biotechnology Co., Ltd.), 0.25% LAP photoinitiator, and 100 ng / mL IRF4 (Abcam, Cambridge, MA, USA, Cat#ab132104) was added to the bioprinting ink. The 3D printer (SUNP Biomaker, SunP (Beijing) Biotechnology Co., Ltd.) was configured with a nozzle temperature of 28°C, a platform temperature of 10°C, a printing speed of 3 mm / s, and UV sterilization. The completed bioprinting ink was placed into a syringe with a needle-sized tip (approximately 350 μm inner diameter), and the printing process was initiated. After printing, the 3D scaffold was placed 2–3 cm away from a blue light source (wavelength 405 nm) and allowed to cross-link and cure for 15 seconds to obtain the composite immune scaffold of this invention.

[0073] Physicochemical property characterization methods

[0074] 1. Scanning electron microscope

[0075] The composite immunoscaffold of this application was placed in a vacuum freeze dryer (Alpha 1-2l LDplus, MartinChrist Gefriertrocknungsanlagen GmbH, Osterode, Germany) for 24 hours to dehydrate. After freeze drying, the sample was embedded in a compound at the optimal cutting temperature and cryo-sectioned into sections of about 100 μm thickness using a low-temperature microtome (Leica CM1950, Leica Microsystems GmbH, Germany). The sections were placed in a vacuum chamber, uniformly coated with a thin layer of gold, and then mounted into a field emission scanning electron microscope for morphological imaging.

[0076] 2. Cell staining:

[0077] Add 200 μl of dimethyl sulfoxide to a test tube containing 200 μg of Calcein-AM powder and dissolve it through a tubing. Return to room temperature using Calcein-AM storage solution and propidium iodide (PI) storage solution. Add 10 μl of Calcein-AM storage solution and 15 μl of PI storage solution to 5 ml of phosphate-buffered saline solution to prepare the working solution. At this point, the Calcein-AM concentration is 2 μmol / L, and the PI concentration is 4.5 μmol / L. After preparing the Calcein-AM and PI staining solutions, wash the cells three times with phosphate-buffered saline solution and add the staining solution directly for staining. Remove the supernatant and add phosphate-buffered saline solution to prepare a cell suspension (105-106 ml). Repeat steps 2 and 3 several times to eliminate esterase activity in the medium. Mix 100 μl of the staining working solution with 200 μl of the cell suspension and incubate at 37°C in a humidified incubator with 5% CO2 for 15 min.

[0078] Pre-cultured scaffolds were irradiated at an excitation wavelength of 490±10 nm, and both yellow-green fluorescent live cells and red fluorescent dead cells were observed. Furthermore, dead cells were observed after irradiation at room temperature (37℃) and an excitation wavelength of 545 nm for 15 min.

[0079] 2. Young's modulus determination

[0080] The Young's modulus of the composite immune scaffold was determined using a dynamic kinetic method at a test temperature of 37℃.

[0081] 3. Rheological property determination

[0082] Oscillatory stress and frequency scans were performed at 37°C using a rotational rheometer (MCR 302, Anton Paar GmbH, Austria).

[0083] 4. Contact Angle Measurement

[0084] Measured using a contact angle tester (OCA15EC, DataPhysics Instruments GmbH, Germany).

[0085] 5. Resistivity Measurement

[0086] Measurements were taken at 37°C using the four-probe method.

[0087] 6. Moisture content determination

[0088] Differential scanning calorimetry and thermogravimetric analysis were used for determination.

[0089] 7. Enzyme-linked immunosorbent assay (ELISA)

[0090] IRF4 standard solutions were prepared at concentrations of 2000 pg / mL, 1000 pg / mL, 500 pg / mL, 250 pg / mL, 125 pg / mL, 62.5 pg / mL, and 31.2 pg / mL, respectively. Equal volumes of these standard solutions (100 μL) and IRF4 sample dilution buffer (100 ng / mL) were slowly added to 96-well plates. At different time points, 100 μL of supernatant was collected and added to a new 96-well plate. The plates were sealed with a gel film and incubated at 37°C for 90 min. After incubation, the liquid was removed from each well, and the wells were dried with absorbent paper. 100 μL of an anti-IRF4 anti-inflammatory ELISA kit (JL19414-48T, Shanghai, China, dilution 1:500) was added to each well, the plate was sealed, and incubated at 37°C for 60 min. After incubation, add 300 μL of PBS buffer to each well and soak for 1 min. Then, pour out the liquid from the wells and wash the wells three times with absorbent paper. Next, add 100 μL of benzoic acid-biotin coagulation reagent (Vectastain). Kit, USA), re-seal the microplate, incubate at 37 °C for 30 min, then wash 5 times. Finally, add 90 μL of 3,3',5,5'-tetramethylbenzidine substrate (Sigma-Aldrich, St. Louis, MO, USA) to each well, seal the plate and incubate in an oven at 37 °C for 30 min. After incubation, add 100 μL of 3,3',5,5'-tetramethylbenzidine stop solution to each well and read the absorbance at 450 nm. The OD (optical density) values of the standard solutions are used to construct a standard curve. The IRF4 concentration under each condition is calculated according to the standard curve and statistical analysis of the IRF4 release at each time point is performed. Use an IL-4 ELISA kit (Abcam, Cat#ab100770) and an IL-6 ELISA kit (Abcam, Cat#ab234570) to quantify the levels of IL-4 and IL-6 in the spinal cord according to the manufacturer's instructions. Measure the OD at a wavelength of 450 nm using an ultraviolet spectrophotometer (Shimadzu, Hong Kong, China, UV-1900i).

[0091] 8. Degradation experiment of composite immune scaffold

[0092] Place the composite immune scaffold in a culture dish and observe continuously for 28 days. Use a stereomicroscope (Leica Microsystems, Wetzlar, Germany, model: M205C) to take pictures and observe.

[0093] 9. Animal research design

[0094] In this invention, 15 rats are used for neurophysiological evaluation. The 15 rats are divided into 5 experimental groups, with 3 rats in each experimental group. In this invention, 15 rats are used for Western blot analysis. The 15 rats are divided into 5 experimental groups, with 3 rats in each experimental group. In this invention, 12 rats are used for macrophage M1 and M2 immunofluorescence staining. The 12 rats are divided into 4 experimental groups, with 3 rats in each experimental group. In this invention, 12 rats are used for neuron immunofluorescence staining, cytokine secretion analysis and immunohistochemical analysis. The 12 rats are divided into 4 experimental groups, with 3 rats in each experimental group. The rats selected in this invention are healthy adult female Sprague-Dawley (SD) rats, weighing 200 - 300 g, purchased from Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China, license number SCXK (Jing) 2016 - 0006). The rats are kept in a specific pathogen-free environment with the following living conditions: temperature 22 °C - 24 °C, humidity 50% - 60%, 12 h light / dark cycle.

[0095] The rats are randomly divided into the following groups:

[0096] a. Sham group (n=6; T10 laminectomy, no spinal cord injury).

[0097] b. PBS group (n=12; T10 spinal cord defect model was prepared and PBS solution (0.3ml) was injected in situ.

[0098] c.3D Scaffold group (n=12; T10 spine, spinal cord defect model prepared and composite immune scaffold transplanted).

[0099] d. IRF4 group (n=12; T10 spinal cord defect model was prepared and IRF4 protein solution (0.3ml, 100ng / ml) was injected in situ).

[0100] e.3D Scaffold@IRF4 group (n=12; T10 spinal cord defect model was established by orthotopic injection of composite immune scaffold loaded with IRF4 (0.3ml, 100ng / ml). n refers to the number of rats.

[0101] Rat were anesthetized by intraperitoneal injection of 0.3% pentobarbital (30 mg / kg) (Sigma-Aldrich). A dorsal incision was made to expose the paraspinal muscles and tendons located on both sides of the spine. The hemispinal muscles extend from the L1 region and connect to the midline via tendons. The midpoint of the intervertebral foramen between T1 and T0 was marked. The intervertebral foramen was increased by gentle abdominal pressure, and the rat's head was stabilized with the thumb and forefinger while the tail was fixed. The scalpel was held in the hand with the blade facing outward, parallel to the marked intervertebral foramen. A precise incision was then made to enter the intervertebral region, and a vertical force was applied to sever the vertebral body, with the scalpel handle tilted at a 30° angle to the uncut side. Complete transection of the spinal cord and loss of hindlimb motor function confirmed successful spinal cord injury.

[0102] In the 3D Scaffold and 3D Scaffold@IRF4 groups, 2 mm long composite immune scaffolds were implanted. The PBS group received an injection of the same volume of PBS solution (0.3 ml) at the lesion site, while the IRF4 group received an injection of IRF4 (0.3 mg, 100 ng / ml). The scaffolds were securely placed between the transverse spinal cord segments. Postoperatively, the incisions were sutured, and the mice were placed in natural light to ensure they remained dry. Postoperative care included daily bladder emptying and administration of antibiotics to prevent infection.

[0103] 10. Behavioral Assessment

[0104] Motor function recovery after spinal cord injury (SCI) is assessed using a variety of methods. For example, the BBB functional score is used to evaluate preoperative motor function recovery, with weekly follow-up assessments (1–8 weeks) postoperatively. Pain sensitivity is measured using the Von Frey test, which involves applying different weights of Von Frey hair to the soles of rats, with a minimum weight of 20g to prevent tissue damage. The CatWalk system (CatWalkXT10.6) (Noldus Information Technology, Wageningen, Netherlands) is used for gait analysis. This system automatically calculates gait parameters, including regularity index, footprint position, and hindlimb stance, to assess behavioral changes after SCI.

[0105] 11. Neurophysiological assessment

[0106] Neurophysiological assessments were performed using standardized electrophysiological methods. Assessment equipment included a stimulator (DS7A dual-channel stimulator, Digitimer Ltd., Wilwing Gardens, UK) and a recording system (NeuroLogSystem, Digitimer Ltd.). Post-operatively, rats were securely positioned in a stereotactic frame to minimize movement during data acquisition.

[0107] Motor evoked potentials (MEPS) involve applying electrical stimulation to the spinal cord nerves while simultaneously recording complex muscle action potentials in the lower limbs. Based on the recorded signals, the integrity and synchronicity of the cortical-myeloid pathway are assessed.

[0108] Somatosensory evoked potentials (SEPS) were induced by applying sensory stimulation to the forelimbs, thereby generating neural responses. Probe electrodes were strategically placed on the sensory pathways to capture the evoked potentials, and the latency and waveform characteristics of these responses were analyzed.

[0109] 12. Measurement of residual urine volume: Manual pressure was applied to the lower abdomen of the experimental rats to induce relaxation of the bladder sphincter, thereby promoting spontaneous urine expulsion through the urethra. The collected urine was placed in calibrated test tubes and its volume was recorded.

[0110] 13. Renal Function Assessment: The rat tail was immersed in a temperature-controlled water bath (Shenzhen Huayang Biotechnology Co., Ltd., Shenzhen, China, Cat#36-0051) (45±1℃) for 5 min. Subsequently, the distal tail was removed 3–5 mm to collect blood. After collection, the incision site was disinfected, and pressure was applied to stop bleeding. The blood volume from each rat sample was limited to 0.3–0.5 ml. Serum was obtained by centrifugation at 3000 rpm for 20 min. Blood urea nitrogen (BUN), serum creatinine, and serum albumin were quantified using a biochemical analyzer (Hitachi, Ltd., Tokyo, Hitachi 7180, Japan).

[0111] 14. Western Blotting: Proteins were extracted from spinal cord tissue, and protein concentrations were determined using BCA assay (Thermo Fisher Scientific). Equal volumes of protein were separated by SDS-PAGE (Merck, Burlington, MA) and transferred to PVDF membranes. Cell membranes were incubated with primary antibodies against JAK1, STAT6, IRF4, IRF5, STAT5, and GAPDH. Bands were then visualized using Thermo Fisher Scientific's ECL kit with a secondary antibody conjugated with horseradish peroxidase, and quantified using ImageJ software. Relevant protein expression was normalized. Primary antibodies include: immune antibody JAK1 (1:200, Abcam, Cat#ab138005), immune antibody STAT6 (1:400, Abcam, Cat#ab251555), immune antibody IRF4 (1:1000, Abcam, Cat#ab315394), immune antibody IRF5 (1:200, Abcam, Cat#ab181553), and immune antibody STAT5 (1:1000, Abcam, Cat#ab32043). Secondary antibodies (all from Abcam) included: Alexa Fluor 568 conjugated to goat anti-immune (1:500, Cat#ab312925), HRP conjugated to goat anti-immune (1:5000, Cat#ab196480), HRP conjugated to goat anti-rat (1:5000, Cat#ab97051), HRP conjugated to goat anti-rat (1:20000, Cat#ab205718), and Alexa Fluor 568 conjugated to goat anti-rabbit (1:500, Cat#ab312939). All samples were incubated overnight at 4°C.

[0112] 15. Immunofluorescence staining

[0113] Spinal cord tissue was collected 8 weeks after SCI, fixed in 4% formaldehyde, and cryo-cut into 10 μm fragments using a cryostat (Leica Microsystems, Wetzlar, Germany, Cat#CM1860). Immunofluorescence staining was used to detect CD206 (anti-labeled, immune, 1:100, Abcam, cat#ab64693), CD86 (pro-inflammatory labeled, immune, 1:200, Abcam, cat#ab53004), β-tubulin (pro-inflammatory labeled, mouse, 1:500, Abcam, cat#ab18207), calmodulin (CAM) (live-cell labeled, Thermo Fisher Scientific, cat#C3099, diluted 1:500), and propidium iodide (dead-cell labeled, 1:1000, Thermo Fisher Scientific, cat#3566). Neural markers (NeuN rabbit, 1:200, Abcam, Cat#ab177487, RRID: AB2532100) were incubated overnight at 4°C. Cell nuclei were counterstained with DAPI (Thermo Fisher Scientific). Tissue sections were visualized using a confocal microscope (OLYMPUS), and the number of neurons and glial cells was counted using ImageJ software.

[0114] 16. Hematoxylin and eosin (H&E) and immunohistochemical staining

[0115] Eight weeks post-surgery, the animals were euthanized using carbon dioxide according to ethical guidelines. Spinal cord segments surrounding the lesion were dissected at 4°C and fixed overnight in 4% paraformaldehyde. The fixed tissue was dehydrated using graded ethanol series, cleaned with xylene, and embedded in paraffin. Sections were cut into 5–10 μm thick fragments using a microscope and mounted on glass slides.

[0116] The integrity of the structures was assessed using hematoxylin and eosin (H&E) staining techniques. Sections were first immersed in hematoxylin (Sigma-Aldrich) staining solution for 5 min, then rinsed with water. Next, the sections were immersed in eosin (Sigma-Aldrich) staining solution for 3 min, and rinsed with tap water. Finally, the sections were dehydrated with a series of ethanol solutions, cleared in xylene, and fixed onto coverslips using a permanent fixative.

[0117] For immunohistochemical staining, sections were dewaxed in xylene, rehydrated with fractionated ethanol, and subjected to heat-induced epitope recovery in citrate buffer (pH 6.0) at 95–98°C for 10–20 min. Endogenous peroxidase activity was blocked with 3% hydrogen peroxide (Sigma-Aldrich) for 10 min, followed by nonspecific binding blockade with 5%–10% normal serum at room temperature for 30 min. Sections were incubated overnight at 4°C. After washing with PBS (Thermo Fisher Scientific) containing primary antibodies against neural markers (NeuN, rabbit, 1:200, Abcam, Cat#ab177487) and glial markers (GFAP, rabbit, 1:500, Abcam, #Cat#ab7200), sections were incubated for 30 min with biotinylated secondary antibodies (anti-rabbit, Abcam, Cambridge, MA, USA) and then developed using horseradish peroxidase (HRP) combined with streptavidin (Abcam, Cambridge, MA, USA) and 3,3'-diaminobenzidine (DAB). Serotonin (Sigma-Aldrich) was used for reverse staining to observe cell nuclei and background. Finally, sections were dehydrated by graded ethanol, cleaned in xylene, and mounted with caps using permanent mounting media.

[0118] The statistical analyses described above were performed using GraphPad Prism 8 (v8.0.2, GraphPad Software, San Diego, CA, www.graphpad.com). For comparisons among multiple groups, one-way ANOVA followed by Tukey's post-hoc test was used. Results are expressed as mean ± standard deviation (SD), and statistical significance was defined as a p-value less than 0.05.

[0119] Molecular dynamics simulation:

[0120] All protein structures used in this application were obtained from UniProt (https: / / www.uniprot.org / ). Complete structures predicted by AlphaFold were selected for subsequent analysis. HDOCK software (https: / / hdock.phys.hust.edu.cn / ) was used for flexible ligand docking simulations of rigid and flexible proteins. Finally, PyMOL software (https: / / pymol.org / ) was used to visualize the molecular docking results.

[0121] The molecular dynamics simulation initially used the molecular docking program of the HDOCK program.

[0122] The docking complexes included IRF5-MyD88, IRF4-MyD88, IRF4-MyD88-IRF5, and IRF4-STAT6, followed by 50 ns molecular dynamics simulations in an aqueous environment. The simulation system used an Amber99SB force field at 25 °C and pressure. The system underwent 100 ps NVT equilibration followed by 100 ps NPT equilibration. A final simulation was performed after the preequilibration phase, lasting 50 ns.

[0123] System 1: Molecular dynamics simulations were performed for 50 ns on the docked IRF4 and MyD88 protein complex in aqueous solution. The system was constructed using GROMACS2021 (https: / / www.gromacs.org / ), consisting of one IRF4 protein molecule, one MyD88 protein molecule, and 103202 water molecules, with the addition of Na. + To balance the charge in the system. (Using...)

[0124] The Amber99SB force field was simulated with a chamber size of 11nm*12nm*11nm, operating at 25°C and standard atmospheric pressure. The simulation was performed using the following steps: energy minimization, 100ps NVT, 100ps NPT, followed by a 50ns simulation.

[0125] System 2: Molecular dynamics simulations of the docked IRF5 and MyD88 protein complex in aqueous solution were performed for 50 ns. The system was constructed using GROMACS2021, consisting of one IRF5 protein molecule, one MyD88 protein molecule, and 82,824 water molecules, with the addition of Na. + To balance the charge in the system. Using an Amber99SB force field, the simulation chamber was set to 7nm × 12nm × 18.5 and operated at 25°C and standard atmospheric pressure. The simulation was run through the following sequence of steps: energy minimization, 100ps NVT, 100ps NPT, followed by a 50ns simulation.

[0126] System 3: Molecular dynamics simulation of the docked IRF4 and STAT6 protein complex in aqueous solution over 50 ns. Constructed using GROMACS2021, it consisted of one IRF4 protein molecule, one STAT6 protein molecule, and 222,179 water molecules, with the addition of Na. + To balance the charge in the system. Using an Amber99SB force field, the simulation chamber was set to 13nm × 17nm × 18 and operated at 25°C and standard atmospheric pressure. The simulation was run through the following sequence of steps: energy minimization, 100ps NVT, 100ps NPT, followed by a 50ns simulation.

[0127] System 4: The docked IRF4 protein, MyD88 protein, and IRF5 protein complex were subjected to 50 ns of molecular dynamics simulation in aqueous solution. The system was constructed using GROMACS 2021 and consisted of 1 IRF4 molecule, 1 MyD88 protein molecule, 1 IRF5 protein molecule, and 260517 water molecules, and Na + was added to balance the charge. The Amber99SB force field was used. The simulation box size was 25.5 nm × 26.5 nm × 25.5 nm, and it was run at 25 °C and standard atmospheric pressure (298.15 K). The simulation was run through the following series of steps: energy minimization, 100 ps NVT, 100 ps NPT, followed by a 50 ns simulation.

[0128] BM-MSCs extraction: BM-MSCs were isolated from the femurs and tibias of 6- to 8-week-old female Sprague-Dawley rats purchased from Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China; license number SCXK (Beijing) 2016-0006). They were cultured in DMEM / F12 medium (Thermo Fisher Scientific, Waltham, MA, USA, catalog number 11320033) containing 10% fetal bovine serum (FBS; Thermo Fisher Scientific, catalog number 10099141C) and 1% penicillin / streptomycin (Gibco, #15070063). The cells were cultured in a 5% CO2 incubator at 37 °C. After 3-5 days, non-adherent cells were removed, and the adherent cells were expanded for subsequent experiments. Microscopic examination showed that BM-MSCs were spindle-shaped, flat, or oval, and the cell nuclei were mainly located in the center or slightly eccentric.

[0129] Biological behavior test of the composite immune scaffold:

[0130] IRF4 regulates macrophage polarization through competitive binding with MyD88 and IRF5. To study the intermolecular interactions, the Uniprot database was first used to obtain the molecular structures of IRF4, IRF5, MyD88, and STAT6. Then, the full-length structures predicted by AlphaFold were selected for further analysis. The molecular structures of IRF4, IRF5, MyD88, and STAT6 are shown in Figure 3 .

[0131] Molecular docking of IRF5 and MyD88 yielded the IRF5-MyD88 complex. In this complex, the core driving force for molecular docking is a network of multiple residue interactions formed by ALA-132, GEN-469, LEU-292, CS-168, and ASC-186, resulting in a total binding free energy of -210.10 kcal / mol. Short-range hydrogen bonds and salt bridges account for approximately 50% of the total binding free energy.

[0132] Molecular docking of IRF4 and MyD88 yielded the IRF4-MyD88 complex and the IRF5-MyD88 complex. In these complexes, the core driving force for molecular docking originates from the interaction network surrounding residues GLN-63, ARG-445, GLU-124, and LYS-127. The binding free energy in this case is -123.66 kcal / mol. Short-range (<0.30 nm) polar and ionic interactions are the main factors influencing binding affinity, while hydrophobic residues such as GLY-427 provide spatial complementarity.

[0133] Molecular docking of IRF4, MyD88, and IRF5 yielded the IRF4-MyD88-IRF5 complex. In this complex, the core driving force for molecular docking originates from an interaction network formed by ASP-283, TRP-465, LEU-471, and MET-426. The binding free energy between IRF4 and MyD88 is -166.59 kcal / mol, with short-range charge and hydrophobic interactions accounting for approximately 60% of the binding energy. The binding energy between IRF5 and MyD88 is weaker, at -17.21 kcal / mol. When IRF4 and IRF5 bind to MyD88 separately, IRF5 exhibits a stronger binding affinity. However, in the IRF4-MyD88-IRF5 complex, the electrostatic interaction between IRF4 and MyD88 weakens the binding of IRF5.

[0134] Molecular docking of IRF4 and STAT6 yielded the IRF4-STAT6 complex. In this complex, the core driving force for molecular docking is a dual-core network involving HIS-233 (through coordination and hydrogen bonding) and PRO-339 (through hydrophobic stacking). The binding free energy here is -216.59 kcal / mol, with short-range interactions accounting for approximately 50% of the total binding energy. The interaction between IRF5 and MyD88 plays a crucial role in guiding giant cell polarization towards the M1 phenotype. Molecular docking analysis showed that IRF4 can bind to MyD88, while in systems containing both IRF4 and IRF5, IRF4 competes with IRF5 for the MyD88 binding site. Furthermore, IRF4 can also interact with STAT6, which may promote M2 giant cell polarization by influencing the JAK / STAT signaling pathway.

[0135] Figure 4A Images of the composite immune scaffold provided in embodiments of this application are shown. Figure 4B SEM images of the composite immune scaffold provided in the embodiments of this application are shown, as follows. Figure 4A and Figure 4B As shown, the composite immune scaffold of this application is about 1 mm in length and has a porous structure on its surface.

[0136] Figure 4C Rheological analysis of the composite immune scaffold provided in the embodiments of this application is shown. Figure 1 , Figure 4D Rheological analysis of the composite immune scaffold provided in the embodiments of this application is shown. Figure 2 ,exist Figure 4C and Figure 4D In this context, G' represents the energy storage modulus, and G” represents the loss modulus, such as... Figure 4C and Figure 4D As shown, the composite immune scaffold of this application has good mechanical properties, which enables the composite immune scaffold to maintain structural integrity when implanted at the site of spinal cord injury, reduce deformation during use, protect the nerve tissue at the site of spinal cord injury, prevent secondary spinal cord injury, and promote the regeneration of the injured tissue.

[0137] Figure 4E The image shows the contact angle measurement results of the composite immune scaffold provided in the embodiments of this application, as shown in the figure. Figure 4EAs shown, the contact angle of the composite immune scaffold in this application is less than 90°, indicating that the composite immune scaffold has good hydrophilicity, enabling the attachment of key extracellular matrix proteins (such as fibronectin and laminin), which are essential for cell adhesion. For osteoblasts and stem cells, the hydrophilic surface of the composite immune scaffold can mimic the polarity of the extracellular matrix, thereby enhancing cell adhesion, spreading, and proliferation. In addition, the hydrophilicity of the composite immune scaffold reduces the resistance to cell contact, thereby accelerating tissue regeneration and repair processes, promoting the diffusion of water molecules, and helping to regulate the sustained release of IRF4 on the composite immune scaffold.

[0138] Figure 4F The drug release curve of the composite immune scaffold provided in the embodiments of this application is shown, as follows: Figure 4F As shown, the IRF4 in this composite immune scaffold is rapidly released, reaching a high concentration within the first 7 days, which coincides well with the acute phase of spinal cord injury. This rapid release is highly beneficial for regulating the inflammatory environment in the acute phase of spinal cord injury. Subsequently, the release rate of IRF4 decreases, but it can still be released continuously for 28 days. Compared to the short half-life of IRF4 released naturally, this application, by using a scaffold to carry IRF4, can significantly prolong the release time of IRF4, ensuring the sustained therapeutic effect of IRF4 protein in the tissues surrounding spinal cord injury, and is beneficial for regulating macrophage polarization over a longer period of application. Figure 4F The values ​​are expressed as mean ± standard deviation.

[0139] BM-BSCs were seeded onto the composite immunoassay scaffold of this application and incubated at 37°C in a 5% carbon dioxide atmosphere. Samples were stained at 7, 14, 21 and 28 minutes using a cytotoxicity kit (#L3224, Thermo Fisher Scientific) with calcein-AM (live cells, green fluorescence) and ethidium homodimer-1 (dead cells, red fluorescence) at room temperature for 30 minutes. Finally, the samples were visualized using a confocal microscope (LSM880, Germany).

[0140] Figure 5 The survival status of BM-MSCs on the composite immune scaffold is shown at days 7, 14, and 28, as follows: Figure 5 As shown, after co-culturing the composite immune scaffold of this application with BM-MSCS for 4 weeks, BM-MSCS (bone marrow-derived mesenchymal stem cells) formed multicellular aggregates within the composite immune scaffold matrix, exhibiting a star-shaped morphology. These structures resemble a network, interacting with neighboring cells through intercellular connections. Figure 5 The detection of very little cell death confirms the high biocompatibility of the composite immune scaffold in this application, which is beneficial for transplantation into the human body.

[0141] Composite immune scaffolds enhance functional recovery after spinal cord injury

[0142] Figure 6A The diagram shows the potential results of rats after limb amputation followed by implantation into the Sham group, PBS group, 3D Scaffold group, IRF4 group, and 3DScaffold@IRF4 group, respectively. Figure 6B The statistical results of potentials in rats after limb amputation followed by implantation in the Sham group, PBS group, 3D Scaffold group, IRF4 group, and 3D Scaffold@IRF4 group are shown. Figure 6A and Figure 6B In this context, MEP represents the motor evoked potential, and SEP represents the somatosensory evoked potential. Figure 6A and Figure 6B As shown, after spinal cord injury, the waveforms and amplitudes of motor and sensory evoked potentials in the PBS group, 3DScaffold group, and IRF4 group were all relatively small. The amplitude of the 3DScaffold@IRF4 group increased, and the waveform was similar to that in normal tissue (i.e., the Sham group), indicating that the 3DScaffold@IRF4 group can promote the recovery of neural conduction potentials.

[0143] Figure 6C The BBB scores of rats after limb amputation and implantation in the PBS group, 3D Scaffold group, IRF4 group, and 3D Scaffold@IRF4 group are shown. Figure 6C As shown, compared with other groups, the 3D Scaffold@IRF4 group showed significantly improved motor function recovery.

[0144] Figure 6D The results of weekly postoperative hindlimb support angle recordings in rats implanted with PBS, 3D Scaffold, IRF4, and 3D Scaffold@IRF4 groups are shown. Figure 6D As shown, compared to other groups, the 3D Scaffold@IRF4 group showed significantly enhanced hind limb support.

[0145] Figure 6E The Von Frey scores of the left hind limb after limb amputation in rats implanted with PBS, 3D Scaffold, IRF4, and 3D Scaffold@IRF4 groups are shown. Figure 6F The Von Frey scores of the right hind limbs implanted after limb amputation in rats in the PBS group, 3DScaffold group, IRF4 group, and 3D Scaffold@IRF4 group are shown. Figure 6E and Figure 6FAs shown, sensory function recovery in the 3D Scaffold@IRF4 group exhibited a delayed but gradual trend, with significant improvement observed starting from the third week post-implantation. Compared to other groups, motor function recovery in the 3D Scaffold@IRF4 group showed an earlier and faster recovery process, with measurable recovery observed as early as the first week. Throughout the observation period, sensory recovery in the 3D Scaffold@IRF4 group was significantly slower than motor recovery.

[0146] Figure 6G The study shows the urine output of rats implanted with PBS, 3D Scaffold, IRF4, and 3D Scaffold@IRF4 at 8 weeks post-operatively. Figure 6F As shown, the 3D Scaffold@IRF4 group alleviated neurogenic bladder symptoms, including urinary retention, over time, thus contributing to the recovery of bladder function.

[0147] Figure 6H The results of blood urea nitrogen and serum creatinine levels in rats after limb amputation and implantation in the PBS group, 3D Scaffold group, IRF4 group, and 3D Scaffold@IRF4 group are shown. Figure 6H As shown, the renal function recovery in the 3D Scaffold@IRF4 group was significantly improved.

[0148] Determination of macrophage polarization type regulation by composite immune scaffold:

[0149] Figure 7A The diagram shows a quantitative analysis of macrophages implanted in the spinal cord injury sites of rats in the PBS group, 3D Scaffold group, IRF4 group, and 3D Scaffold@IRF4 group 8 weeks later. Figure 7A In the image, CD86 represents macrophages, DAPI represents the image of CD86 cells stained with DAOI (4′,6-diamino-2-phenylindole), and Merge represents the image of CD86 cells after Merge image processing. Figure 7A As shown, a large number of pro-inflammatory macrophages were observed in the spinal cord tissue of the PBS group and the 3D Scaffold group, while the number of pro-inflammatory macrophages in the spinal cord tissue of the IRF4 group was reduced, and the number of pro-inflammatory macrophages in the spinal cord tissue of the 3D Scaffold@IRF4 group was extremely low.

[0150] Figure 7B The diagram shows the quantitative analysis of anti-inflammatory macrophages 8 weeks after implantation into the spinal cord injury sites of rats in the PBS group, 3D Scaffold group, IRF4 group, and 3D Scaffold@IRF4 group. Figure 7BIn the image, CD206 represents anti-inflammatory macrophages, DAPI represents the image of CD206 cells stained with DAPI (4′,6-diamino-2-phenylindole), and Merge represents the image of CD206 cells after Merge image processing. Figure 7B As shown, few anti-inflammatory macrophages were detected in the spinal cord of the PBS and 3D Scaffold groups, the IRF4 group showed an increased number of anti-inflammatory cells, and the number of anti-inflammatory giant cells was significantly increased in the 3D Scaffold@IRF4 group.

[0151] Figure 7C The results of quantitative analysis of M1 macrophages, M2 macrophages, and cytokines IL-6 and IL-4 were shown 8 weeks after implantation into the spinal cord injury sites of rats in the PBS group, 3D Scaffold group, IRF4 group, and 3D Scaffold@IRF4 group. Figure 7C As shown, compared with the PBS group, there were significant differences in macrophage subtypes in the 3D Scaffold group and the IRF4 group. Further investigation of macrophage secretory function revealed no significant difference between the 3D Scaffold group and the IRF4 group. In the 3D Scaffold@IRF4 group, the secretion of the pro-cytokine IL-6 was significantly reduced, while the secretion of the anti-inflammatory cytokine I-4 was significantly increased, which can promote the transformation of macrophages from the M1 phenotype to the M2 phenotype. This transformation is conducive to the formation of an inflammatory environment conducive to nerve regeneration at the site of spinal cord injury. Therefore, it can be determined that the 3D Scaffold@IRF4 group can regulate macrophage polarization to the M2 phenotype.

[0152] Assay for nerve cell regeneration and tissue recovery:

[0153] Figure 8A The image shows H&E staining patterns of the PBS group, 3D Scaffold group, IRF4 group, and 3D Scaffold@IRF4 group after implantation into the spinal cord injury site of rats. Figure 8A As shown, in the PBS group, after spinal cord injury in rats, complete spinal cord transection resulted in localized fibrous scarring with minimal spinal cord tissue. The 3D Scaffold group preserved the entire spinal cord structure; however, due to scaffold degradation, regenerated tissue was almost undetectable. The IRF4 group not only failed to maintain spinal cord structure but also exhibited fragmented regenerated tissue, failing to effectively restore neurological function. The 3D Scaffold@IRF4 group not only preserved the spatial structure of the spinal cord but also promoted integration with host tissue, facilitating the growth of regenerated tissue into a composite immune scaffold.

[0154] Immunohistochemical staining was performed to further assess the condition of neurons and scar tissue. Figure 8BThe images show NeuN immunostaining images of spinal cord neurons implanted in the spinal cord injury sites of rats in the PBS group, 3D Scaffold group, IRF4 group, and 3D Scaffold@IRF4 group. Figure 8C The image shows GFAP immunostaining images of astrocytes in the spinal cord tissue of rats implanted in the PBS group, 3D Scaffold group, IRF4 group, and 3D Scaffold@IRF4 group at the site of spinal cord injury, as shown. Figure 8B and Figure 8C As shown, neurons were almost invisible in the PBS group, while astrocytes were significantly aggregated. No visible neurons were observed in the 3D Scaffold group, although the number of astrocytes was reduced due to the scaffold's filling effect. A small number of neurons and astrocytes were detected in the IRF4 group, while the 3D Scaffold@IRF4 group showed significant neuronal presence and almost no astrocytes. This indicates that the composite immune scaffold can promote neuronal regeneration and reduce glial scar formation.

[0155] Figure 9 The images show representative immunofluorescence staining of β-III tubulin and NeuN (neuron-specific enolase) after implantation into the spinal cord injury sites of rats in the PBS group, 3D Scaffold group, IRF4 group, and 3D Scaffold@IRF4 group. Figure 9 In the diagram, green represents β-III tubulin, red represents NeuN, and each group of cell nuclei is labeled with DAPI (blue). Figure 9 As shown, almost no neurons and axons were observed in the PBS and 3D Scaffold groups, while a very small number of neurons and regenerating axons were observed in the IRF4 group. The 3D Scaffold@IRF4 group significantly promoted the regeneration of neurons and axons, and there were statistically significant differences between the groups.

[0156] Molecular mechanism research and experimental verification:

[0157] Figure 10A The molecular dynamics simulation results of the IRF4-MyD88 complex are shown, such as... Figure 10AAs shown, in the IRF4-MyD88 complex, the total binding free energy (BFE) is -123.66 kcal / mol, and the binding energy remains relatively stable (-260 ± 5 kcal / mol) throughout the simulation, exhibiting minimal fluctuations in the initial 10 ns (ABFE ≤ 3 kcal / mol). This indicates that the dimer complex possesses high binding stability. The root mean square deviation (RMSD) fluctuates between 0.18 and 0.22 nm, indicating that the acceptor-ligand interface conformation is highly conserved. The solvent accessible surface area (SASA) exhibits periodic fluctuations, possibly due to minor conformational changes in local residues, particularly in the TIR domain of MyD88.

[0158] Figure 10B The molecular dynamics simulation results of the IRF5-MyD88 complex are shown, such as... Figure 10B As shown, the total binding free energy (BFE) in the IRF5-MyD88 complex is -210.10 kcal / mol. Initially, the BFE decreases to -255 kcal / mol (ABFE ≈ -5 kcal / mol), then gradually increases to -245 kcal / mol, reflecting a decrease in binding affinity between the ligand and acceptor during simulations. The RMSD value fluctuates between 2.0 and 2.8 Å, indicating greater flexibility of the IRF5-MyD88 interface. The SASA continuously increases (2.8–3.5 nm), possibly due to the exposure of flexible side chains, such as aromatic rings, in IRF5 to the solvent environment.

[0159] Figure 10C The molecular dynamics simulation results of the IRF4-MyD88-IRF5 complex are shown, such as... Figure 10C As shown, in the IRF4-MyD88-IRF5 ternary complex, the total BFE of IRF4-MyD88 is -166.59 kcal / mol, while that of IRF5-MyD88 is -17.21 kcal / mol. Throughout the simulation, the BFE remained stable at -290 ± 3 kcal / mol with negligible fluctuations (ABFE < 2 kcal / mol), indicating that the ternary synergistic effect significantly enhanced the binding stability. The RMSD value remained in the range of 0.12–0.15 nm, significantly lower than that in the dimer system, indicating the formation of a robust core structure through hydrophobic stacking and bond network. The SASA decreased to 2.0–2.4 nm, possibly because a tight hydrophobic barrier was established at the ternary interface, thus limiting the penetration of solvent molecules.

[0160] Figure 10D The molecular dynamics simulation results of the IRF4-STAT6 complex are shown, such as... Figure 10DAs shown, the BFE value in the IRF4-STAT6 complex is -216.59 kcal / mol. During the simulation, the BFE initially decreased to -270 kcal / mol and then gradually increased to -250 kcal / mol (ABFE was 20 kcal / mol), indicating a significant decrease in binding affinity during the simulation. The RMSD values ​​showed significant fluctuations, particularly in the SH2 domain region of STAT6 (residues 400-500), where conformational changes were more pronounced. The SASA exhibited a bimodal distribution, which may correspond to the periodic exposure or concealment of phosphorylation sites on STAT6.

[0161] In summary, the BFE of IRF4 with MyD88 is -123.66 kcal / mol, while IRF5 exhibits a stronger binding affinity for MyD88 (-210.10 kcal / mol). Further analysis of the IRF4-MyD88-IRF5 system reveals that IRF4 has a significantly higher binding affinity for MyD88 (-166.59 kcal / mol) compared to IRF5 (-17.21 kcal / mol), indicating that IRF4 more effectively competes for binding with MyD88. Therefore, it can be confirmed that IRF4 can replace IRF5 from its binding site, favoring M2 polarization. Furthermore, electrostatic interactions and van der Waals forces stabilize the IRF4-MyD88 binding, with electrostatic interactions contributing the most significant amount to the stability of the complex. In the IRF4-STAT6 complex, the BFE value was -216.59 kcal / mol, indicating that IRF4 and STAT6 had already bound, but the binding was through van der Waals interactions. Thus, the interaction between IRF4 and STAT6 may enhance the polarization of M2 macrophages by regulating the JAK / STAT pathway.

[0162] Figure 10E Western blot analysis of JAK1, STAT6, and IRF4 protein expression in different groups. Figure 10F The results of quantitative analysis of JAK1, STAT6 and IRF4 protein expression in different groups are shown. Figure 10G Western blot analysis of IRF5 and STAT5 protein expression in different groups. Figure 10H The figure shows the results of quantitative analysis of IRF5 and STAT5 protein expression in different groups. Figure 10I The relative expression levels of components JAK1, STAT6, IRF4, IRF5, and STAT5 were compared. Data are expressed as mean ± SD(n-3). Figures 10E to 10IAs shown, compared to other groups, the expression of IRF4, JAK1, and STAT6 was significantly increased in the 3D Scaffold@IRF4 group, while the expression of IRF5 and STAT5 was significantly decreased in the 3DScaffold@IRF4 group. This indicates a competitive interaction between IRF4 and IRF5 in their competition for the MyD88 binding site. Specifically, IRF4 reduces the expression of pro-inflammatory proteins by competitively inhibiting the binding of IRF5 to MyD88, thereby suppressing the polarization of M1 macrophages. Simultaneously, IRF4 promotes the polarization of M2 macrophages by activating the JAK1 / STAT6 pathway. Through these mechanisms, IRF4 regulates the activity of inflammatory cells, modulates the secretion of inflammatory factors, and improves the inflammatory microenvironment, thereby promoting neurogenesis.

[0163] The applicant declares that the above description is only a specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention fall within the protection and disclosure scope of the present invention.

Claims

1. A composite immune scaffold for promoting spinal cord injury repair, characterized in that, The composite immune scaffold is formed by 3D printing technology. The composite immune scaffold includes at least two matrix layers and at least one support layer. The support layer is located between the at least two matrix layers. IRF4 protein is distributed inside and / or on the surface of the support layer and the matrix layer.

2. The composite immune scaffold according to claim 1, characterized in that, The concentration of the IRF4 protein was 100 ng / ml.

3. The composite immune scaffold according to claim 1, characterized in that, The materials used to print the composite immune scaffold include gelatin hydrogel containing grafting functional groups, a blue light initiator, and IRF4 protein.

4. The composite immune scaffold according to claim 1, characterized in that, The composite immune scaffold includes at least one of the following features: (1) The support layer includes a plurality of support units arranged at intervals, and pores are formed between the plurality of support units; (2) The contact angle of the composite immune scaffold is less than 90°.

5. The application of a composite immune scaffold for promoting spinal cord injury repair as described in any one of claims 1 to 4, characterized in that, The composite immune scaffold can inhibit M1 macrophage polarization and promote M2 macrophage polarization.

6. The application according to claim 5, characterized in that, The composite immune scaffold can promote the secretion of anti-inflammatory factor IL-4, inhibit the secretion of pro-inflammatory factor IL-6, and regulate the immune microenvironment after spinal cord injury.

7. The application according to claim 5 or 6, characterized in that, After the composite immune scaffold is implanted into the spinal cord injury site, the IRF4 protein can be continuously released for 28 days.

8. The application according to any one of claims 5 to 7, characterized in that, After the composite immune scaffold is implanted into the site of spinal cord injury, the composite immune scaffold can preserve the spatial structure of the spinal cord and promote the integration of the composite immune scaffold with the host tissue within the spinal cord.

9. The application according to any one of claims 5 to 8, characterized in that, After the composite immune scaffold is implanted into the spinal cord injury site, it can promote the effective recovery of motor, sensory, urinary functions and neurophysiological conduction after spinal cord injury.

10. The application according to any one of claims 5 to 9, characterized in that, After the composite immune scaffold is implanted into the site of spinal cord injury, it can promote the regeneration of neurons and axons and reduce the formation of glial scars.

Images