Extracellular matrix-cell complex for inhibiting intervertebral disc degeneration and preparation method and application thereof

Abstract

The present application relates to a kind of extracellular matrix-cell complex for inhibiting intervertebral disc degeneration and its preparation method and application, the extracellular matrix-cell complex for inhibiting intervertebral disc degeneration includes modified cell and the biomimetic extracellular matrix nanofiber constructed on the surface of modified cell.The present application is creatively modified to implant cell in cell therapy, constructs artificial extracellular matrix with nanofiber characteristics in situ on the surface of cell, the complex is implanted into nucleus pulposus by cell injection therapy, can induce cell rest, so that implanted cell is better adapted to the intracellular environment of nucleus pulposus anoxia.The artificial extracellular matrix can be long-term targeted to cell surface, significantly prolongs the action time, and different modified cells can be selected according to actual needs, so that intervertebral disc degeneration cell therapy can be better realized, with wide application prospect.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology and relates to an extracellular matrix-cell complex that inhibits intervertebral disc degeneration, its preparation method and application, specifically to an extracellular matrix-cell complex that inhibits intervertebral disc degeneration, its preparation method and its application in the preparation of pharmaceutical preparations or intervertebral disc implant materials with the effect of inhibiting intervertebral disc degeneration. Background Technology

[0002] Treatment of intervertebral disc degeneration is a major clinical challenge, especially for lower back pain caused by lumbar disc degeneration. Currently, conservative treatment for disc degeneration only provides symptom relief, while surgical interventions to remove the source of pain (such as vertebral fusion) are the ultimate intervention, but they cannot cure the pathological process of disc degeneration. Furthermore, segmental fusion of degenerated discs increases the incidence of adjacent vertebral diseases, and simply removing the herniated nucleus pulposus can exacerbate the degeneration process.

[0003] Intradiscal cell injection therapy to restore intervertebral disc homeostasis is a promising treatment method. For example, CN111067920A reports a formulation and preparation method for treating lumbar intervertebral disc degeneration. The formulation contains mesenchymal stem cells, which are induced and cultured in a conditioned medium containing chondrocytes, which promotes the differentiation of mesenchymal stem cells. The mesenchymal stem cells are encapsulated in hydrogel, which enhances their survival rate and viability in vivo. A thermostatic syringe for the above-mentioned formulation for treating lumbar intervertebral disc degeneration is also provided. The syringe tube is filled with a thermostatic material to maintain the temperature of the contents, which helps maintain cell activity and reduces the body's stress response.

[0004] For example, CN108014339A reports a method and composition for differentiating cells, including human fibroblasts, into chondrocyte-like cells via in vivo mechanical tension. In a specific aspect, fibroblasts are delivered to joints, such as intervertebral discs, where they subsequently differentiate into chondrocyte-like cells to treat cartilage dysfunction, including, for example, repairing degenerated intervertebral discs. Fibroblasts that do not differentiate into chondrocytes due to cell localization or other biomechanical and biochemical microenvironmental factors can produce fibrous matrix molecules to aid in tissue repair and regeneration of the nucleus pulposus and annulus fibrosus.

[0005] However, current cell therapies cannot guarantee a suitable nutrient-metabolite environment. Intervertebral disc regeneration can abnormally activate cellular anabolic activities, leading to increased nutrient demands and ultimately cell death, making the therapy's effectiveness uncertain. Therefore, maintaining low metabolic activity in implanted cells is crucial for cell therapy of intervertebral disc degeneration. Summary of the Invention

[0006] To address the shortcomings of existing technologies, the present invention aims to provide an extracellular matrix-cell complex for inhibiting intervertebral disc degeneration, its preparation method, and its application. Specifically, it provides an extracellular matrix-cell complex for inhibiting intervertebral disc degeneration, its preparation method, and its application in the preparation of pharmaceutical preparations or intervertebral disc implant materials with the effect of inhibiting intervertebral disc degeneration.

[0007] To achieve this objective, the present invention adopts the following technical solution:

[0008] In a first aspect, the present invention provides an extracellular matrix-cell complex for inhibiting intervertebral disc degeneration, the extracellular matrix-cell complex for inhibiting intervertebral disc degeneration comprising modified cells and biomimetic extracellular matrix nanofibers constructed on the surface of the modified cells.

[0009] The modified cells are selected from any one or a combination of at least two of the following: nucleus pulposus cells, nucleus pulposus stem cells, bone marrow mesenchymal stem cells, fibroblasts, or embryonic stem cells.

[0010] As is known to those skilled in the art, nucleus pulposus cells, nucleus pulposus stem cells, bone marrow mesenchymal stem cells, fibroblasts, or embryonic stem cells are commonly used cell types in cell therapy for lumbar intervertebral disc degenerative diseases. However, a drawback is that these cells have difficulty adapting to the nutrient-deficient internal environment of the nucleus pulposus, resulting in poor therapeutic effects. Therefore, this invention overcomes the above-mentioned drawbacks by using a biomimetic extracellular matrix with deformable nanofibers to mediate the quiescent state of implanted cells, thereby achieving effective cell therapy.

[0011] This invention creatively develops an extracellular matrix-cell complex for inhibiting intervertebral disc degeneration. Specifically, it modifies cells for cell therapy implantation, constructing an artificial extracellular matrix with nanofiber characteristics on the cell surface in situ. This complex is implanted into the nucleus pulposus via cell injection therapy, inducing cell quiescence and allowing the implanted cells to better adapt to the nutrient-deficient internal environment of the nucleus pulposus. This artificial extracellular matrix can target the cell surface for extended periods, significantly prolonging its effect. Furthermore, different modified cells can be selected according to specific needs, enabling better cell therapy for intervertebral disc degeneration and demonstrating broad application prospects.

[0012] Preferably, the biomimetic extracellular matrix nanofibers are obtained by the self-assembly of nanopeptide drugs and their interaction with the modified cells through ligand-receptor interactions, resulting in deformation.

[0013] The nanopeptide drug comprises sequentially connected hydrophobic units, self-assembly units, and targeting units.

[0014] The nanopeptide drugs described above have good biocompatibility after self-assembly. They can target and bind to receptors on the cell surface, and then transform into nanofibers that attach to the cell membrane surface, thereby realizing the construction of an artificial extracellular matrix.

[0015] Preferably, the hydrophobic unit is linoleic acid.

[0016] Preferably, the self-assembly unit is a polypeptide sequence including FFVLK.

[0017] Preferably, the targeting unit is a CXCR1 ligand whose sequence includes FKPHFPKSYTKICQ.

[0018] Nanopeptide drugs targeting CXCR1 ligands self-assemble to construct an artificial extracellular matrix with nanofiber characteristics on the cell surface. This competitively inhibits the effect of CXCL8 on cells, thereby inducing cell quiescence and enabling implanted cells to better adapt to the nutrient-deficient internal environment of the nucleus pulposus.

[0019] In a second aspect, the present invention provides a method for preparing an extracellular matrix-cell complex for inhibiting intervertebral disc degeneration according to the first aspect, the method comprising:

[0020] Nanopeptide drugs are self-assembled to form nanoparticles, which are then mixed with modified cells and co-incubated to obtain the extracellular matrix-cell complex that inhibits intervertebral disc degeneration.

[0021] The method for preparing the extracellular matrix-cell complex involved in this invention is simple and easy to operate, making it very suitable for large-scale industrial production and possessing practical value.

[0022] Preferably, the nanoparticles formed by the self-assembly of the nanopeptide drug have a particle size of 10-60 nm, such as 10 nm, 20 nm, 30 nm, 40 nm, 50 nm or 60 nm.

[0023] Preferably, in the co-incubation system, the concentration of the nanoparticles is 5-100 μg / mL, more preferably 5-20 μg / mL; the concentration of the modified cells is (2 × 10⁻⁶) / mL. 6 -4×10 6 ) / mL.

[0024] The 5-100 μg / mL can be, for example, 5 μg / mL, 10 μg / mL, 15 μg / mL, 20 μg / mL, 30 μg / mL, 40 μg / mL, 50 μg / mL, 60 μg / mL, 80 μg / mL, 100 μg / mL, etc.

[0025] The (2×10) 6 -4×10 6 For example, 2 × 10⁻⁶ / mL could be used. 6 / mL, 2.5×10 6 / mL, 3×10 6 / mL, 3.5×106 / mL, 4×10 6 / mL, etc.

[0026] Other specific point values ​​within the range of the above values ​​can be selected, and will not be elaborated on here.

[0027] Preferably, the nanopeptide drug is prepared by solid-phase synthesis.

[0028] Preferably, the co-incubation is carried out at 35-39°C for 1-4 hours.

[0029] The 35-39℃ range can be, for example, 35℃, 36℃, 37℃, 38℃, 39℃, etc.

[0030] The 1-4h can be, for example, 1h, 1.5h, 2h, 2.5h, 3h, 3.5h, 4h, etc.

[0031] Other specific point values ​​within the range of the above values ​​can be selected, and will not be elaborated on here.

[0032] Thirdly, the present invention provides the use of the extracellular matrix-cell complex according to the first aspect for inhibiting intervertebral disc degeneration in the preparation of pharmaceutical formulations or intervertebral disc implant materials having the effect of inhibiting intervertebral disc degeneration.

[0033] Compared with the prior art, the present invention has the following beneficial effects:

[0034] This invention creatively develops an extracellular matrix-cell complex for inhibiting intervertebral disc degeneration. It involves modifying cells for cell therapy implantation, such as nucleus pulposus cells, nucleus pulposus stem cells, bone marrow mesenchymal stem cells, fibroblasts, or embryonic stem cells, to construct an artificial extracellular matrix with nanofiber characteristics on the cell surface in situ. This complex is implanted into the nucleus pulposus via cell injection therapy, inducing cell quiescence and allowing the implanted cells to better adapt to the nutrient-deficient internal environment of the nucleus pulposus. This artificial extracellular matrix can target the cell surface for a long period, significantly prolonging the duration of action. Furthermore, different modified cells can be selected according to actual needs, thus better achieving cell therapy for intervertebral disc degeneration, and it has broad application prospects. Attached Figure Description

[0035] Figure 1 These are transmission electron microscope images of two nanopeptide drugs, namely the preparation example and the comparative preparation example, after being mixed with CXCR1 protein.

[0036] Figure 2 Fourier transform infrared spectra and circular dichroism chromatograms of the two nanopeptide drugs in the preparation example and the comparative preparation example after being mixed with CXCR1 protein;

[0037] Figure 3The graph shows the cytotoxicity results of two nanopeptide drugs: the prepared example and the comparative prepared example.

[0038] Figure 4 This is a fluorescence colocalization map of self-assembled particles formed from two nanopeptide drugs, namely the prepared example and the comparative prepared example, on the surface of nucleus pulposus stem cells (NPSCs).

[0039] Figure 5 These are scanning electron microscope images of self-assembled particles formed from two nanopeptide drugs, namely the prepared example and the comparative prepared example, on the surface of nucleus pulposus stem cells (NPSCs, P3) and lens epithelial cells (LECs, P3).

[0040] Figure 6 This is a flow cytometry image of a prepared nanopeptide drug acting on nucleus pulposus stem cells (NPSC);

[0041] Figure 7 This is a flow cytometry quantification of G0 phase cells of a prepared nanopeptide drug acting on nucleus pulposus stem cells (NPSC);

[0042] Figure 8 This is a staining image of β-galactosidase (SA-β-gal) on the action of a nanopeptide drug on nucleus pulposus stem cells (NPSC).

[0043] Figure 9 This is a positive staining quantitative map of the effect of prepared nanopeptide drugs on nucleus pulposus stem cells (NPSC);

[0044] Figure 10 These are in vivo assessment immunofluorescence slide images (NPSCs group and complex group; 3 weeks post-surgery);

[0045] Figure 11 This is an in vivo assessment of immunofluorescence tissue section quantitative analysis (NPSCs group and complex group; 3 weeks post-surgery);

[0046] Figure 12 These are in vivo assessment immunofluorescence slide images (complex group; 3 weeks, 4 weeks, and 5 weeks post-surgery);

[0047] Figure 13 This is an in vivo assessment of immunofluorescence section quantitative analysis (complex genome; 3 weeks, 4 weeks, and 5 weeks post-surgery);

[0048] Figure 14 This is a transmission electron microscopy (TEM) image of the in vivo assessment of biological samples.

[0049] Figure 15 This is an image of a tissue section stained with H&E for in vivo assessment.

[0050] Figure 16 This is a histological scoring chart of H&E-stained tissue sections used for in vivo assessment.

[0051] Figure 17 This is an image of a tissue section stained with safranin and fast green for in vivo assessment.

[0052] Figure 18 This is a histological scoring chart for safranin-fast green stained tissue sections used in vivo for assessment.

[0053] Figure 19 It is an in vivo assessment of small animal MRI scans;

[0054] Figure 20 It is a quantitative Pfirrmann grading of coccygeal intervertebral disc degeneration in vivo;

[0055] Figure 21 This is a quantitative analysis diagram of the nucleus pulposus area of ​​the coccygeal intervertebral disc in vivo;

[0056] Figure 22 This is an immunostaining slide image for in vivo assessment of p-mTOR;

[0057] Figure 23 This is a quantitative analysis of the average fluorescence intensity of DAPI in immunostained sections of p-mTOR in vivo.

[0058] Figure 24 This is a graph showing the quantitative analysis of the average fluorescence intensity ratio of p-mTOR / DAPI in immunostained sections for in vivo assessment of p-mTOR. Detailed Implementation

[0059] The technical solution of the present invention will be further illustrated below through specific embodiments. Those skilled in the art should understand that the embodiments described are merely illustrative of the present invention and should not be construed as limiting the invention in any way.

[0060] The SD rats and nucleus pulposus stem cells used in the following experiments were obtained from the Second Affiliated Hospital of Army Medical University (Xinqiao Hospital).

[0061] The culture medium involved in the following experiments is DMEM medium (purchased from Invitrogen).

[0062] Preparation example (molecule 1)

[0063] This preparation example provides a nanopeptide drug, linoleic acid-FFVLK-FKPHFPKSYTKICQ, whose molecular structure is shown below:

[0064]

[0065] The preparation method is as follows: it is synthesized by solid-phase synthesis according to the above amino acid sequence.

[0066] Comparative preparation example (molecule 2)

[0067] This comparative preparation example provides a nanopeptide drug, linoleic acid-GGAAK-FKPHFPKSYTKIC Q, whose molecular structure is shown below:

[0068]

[0069] The preparation method is as follows: it is synthesized by solid-phase synthesis according to the above amino acid sequence.

[0070] Example 1

[0071] This embodiment investigates the deformation behavior of two nanopeptide drugs (prepared example and comparative preparation example) after being mixed with CXCR1 protein:

[0072] Both the prepared and comparative nanopeptide drugs were prepared by mixing DMSO as solvent to prepare a stock solution with a concentration of 50 μg / μL. This stock solution was then mixed with PBS for self-assembly to obtain a nanoparticle suspension with a concentration of 10 μg / mL. Finally, this suspension was mixed with CXCR1 protein solution (solvent: PBS, obtained by dissolving CXCR1 protein powder in PBS to obtain a protein solution with a concentration of 2.6 μg / mL) and co-incubated at 37°C. Transmission electron microscopy was performed after 0, 1, and 2 days. A control group without CXCR1 protein was also included.

[0073] The results are as follows Figure 1 As shown in the figure (scale bar 200nm), it can be seen that the nanopeptide drug involved in this invention can be transformed from nanoparticles into nanofibers under the action of CXCR1 protein, while the comparative preparation example did not undergo deformation under the action of CXCR1 protein.

[0074] Example 2

[0075] This embodiment explores the deformation patterns of two nanopeptide drugs (prepared example and comparative preparation example) after being mixed with CXCR1 protein:

[0076] Both the prepared and comparative nanopeptide drugs were prepared by mixing DMSO to form a stock solution with a concentration of 50 μg / μL. This stock solution was then mixed with PBS for self-assembly to obtain a nanoparticle suspension with a concentration of 10 μg / mL. Finally, this suspension was mixed with a CXCR1 protein solution (PBS as the solvent, obtained by dissolving CXCR1 protein powder in PBS to obtain a protein solution with a concentration of 2.6 μg / mL) and co-incubated at 37°C. Fourier transform infrared spectroscopy and circular dichroism spectroscopy were performed after 0 and 2 days. A control group without CXCR1 protein was also included.

[0077] The results are as follows Figure 2As shown in the figure, the self-assembled nanoparticles of the nanopeptide drug involved in this invention undergo self-assembly deformation in the form of β-sheet after interacting with CXCR1 protein. The control group or comparative preparation does not undergo β-sheet changes after interacting with CXCR1 protein.

[0078] Example 3

[0079] This embodiment investigates the cytotoxicity of two nanopeptide drugs: a prepared example and a comparative prepared example.

[0080] Intervertebral disc nucleus pulposus stem cells (NPSCs, P3) and lens epithelial cells (LECs, P3) from SD rats were cultured in basal medium (DMEM) containing 10% fetal bovine serum and incubated overnight at 37°C in 5% CO2. After cell confluence, they were transferred to test tubes for concentration. Cells were then counted and the concentration adjusted to 1 × 10⁻⁶ cells / mL. 4 / cm 2 Cells were then transferred to 96-well cell culture plates, with 100 μL of cells in each well. These plates were then placed in an incubator at 37°C containing 5% carbon dioxide. Once the cells adhered to the plates, stock solutions of the two nanopeptide drugs from both the preparation and control examples were prepared using DMSO as solvent, each at a concentration of 50 μg / μL. These stock solutions were then diluted with culture medium to prepare drug solutions of 5 μg / mL, 10 μg / mL, 50 μg / mL, or 100 μg / mL, respectively. The solutions were then replaced with the original culture medium in the wells, and the plates were incubated for another 24 hours. The culture medium was then removed, and CCK-8 solution diluted 1:9 (v / v) with the culture medium was added. Finally, the absorbance of the samples at 450 nm was measured using a multi-mode microplate detection system (EnSpire™, PE, USA) to calculate cell viability for each group. Three independent measurements were performed, and the average values ​​were calculated.

[0081] The results are as follows Figure 3 As shown in the figure, the two nanopeptide drugs in the preparation example and the comparative preparation example of the present invention showed no obvious cytotoxicity to NPSCs and LECs at 10 μg / mL. Preferably, 10 μg / mL is the optimal administration concentration.

[0082] Example 4

[0083] This embodiment investigates the enrichment of self-assembled particles formed from two types of nanopeptide drugs, namely the prepared example and the comparative prepared example, on the surface of nucleus pulposus stem cells (NPSCs):

[0084] Two nanopeptide drugs, a Cy5 fluorescent molecular labeling preparation example and a comparative preparation example, were prepared using existing techniques in the art. A stock solution with a concentration of 50 μg / μL was prepared by mixing DMSO as the solvent and then mixing it with culture medium for self-assembly to obtain a nanoparticle suspension with a concentration of 10 μg / mL. The suspension was then co-incubated with NPSCs (multiple CXCR1 receptors) and LECs (oligo-CXCR1 receptors) at 37°C for 1 h, 2 h, and 4 h, respectively, and the enrichment of fluorescent molecules on the cell membrane surface was observed.

[0085] The results are as follows Figure 4 As shown in the figure (scale bar: 20 μm), compared with LECs containing oligo-CXCR1 receptors, the prepared nanopeptide drug self-assembled particles can accumulate on NPSCs with multiple CXCR1 receptors on the membrane. In contrast, the prepared nanopeptide drug self-assembled particles do not accumulate on any cell membrane and are instead endocytosed. The results indicate that the prepared nanopeptide drug self-assembled particles can target CXCR1 receptors on NPSCs. Unlike the control, the different assembly effect allows the prepared nanopeptide drug self-assembled particles to surround the cell membrane without endocytosis, thereby achieving the formation of an extracellular matrix-cell composition.

[0086] Example 5

[0087] This embodiment observes the morphology of self-assembled particles formed from two types of nanopeptide drugs in the preparation example and the comparative preparation example on the surface of nucleus pulposus stem cells (NPSCs, P3) and lens epithelial cells (LECs, P3):

[0088] NPSCs or LECs were cultured on silicon wafers, respectively. Two nanopeptide drugs (10 μg / mL, DMSO:medium = 1:99, V / V) were co-incubated with NPSCs or LECs at 37°C for 4 h. The co-cultured cells were fixed with paraformaldehyde for 1 h and then further dehydrated. Finally, the cells were coated with gold and observed using a JC-Zeiss scanning electron microscope (Zeiss, Germany).

[0089] The results are as follows Figure 5 As shown in the figure (scale bar: 10 μm; magnified view: 2 μm), it can be seen that the prepared nanopeptide drug self-assembled particles can generate nanofibers on NPSCs and cover the membrane, while a small number of fibers are generated on LECs of oligo-CXCR1 receptors. In contrast, the prepared nanopeptide drug self-assembled particles do not show fibrous structures on NPSCs and LECs.

[0090] Example 6

[0091] This example examines the effect of prepared nanopeptide drugs on the cell cycle of nucleus pulposus stem cells (NPSCs):

[0092] Serum-free culture can induce nucleus pulposus stem cells to enter the quiescent phase, and therefore serves as a positive control. The specific method is as follows: NPSCs were incubated in conventional medium (DMEM medium containing 10% fetal bovine serum), a suspension of prepared nanopeptide drug self-assembled particles (self-assembled particles + DMEM medium containing 10% fetal bovine serum, final particle concentration 10 μg / mL), and serum-free medium (DMEM medium without fetal bovine serum) at 37°C for 48 h in an incubator containing 5% CO2. Cells were collected, the medium was removed, and they were fixed with 70% ethanol. After washing the cells three times with PBS, they were stained with PI (BDpharingen, China) and anti-Ki67 antibody (AF-488 conjugated to Ki67 (D3B5) antibody, #11882, Cell Signaling Technology, China). NPSCs treated with normal medium served as a control, stained with isotype antibody (AF-488 conjugated rabbit IgG, ab199091, Abcam, China). The cells were further analyzed by flow cytometry (Gallios, USA).

[0093] Streaming results as follows Figure 6 As shown, the quantitative statistical results for period G0 are as follows: Figure 7 As shown in the figure, compared with cells incubated in normal culture medium, both the prepared nanopeptide drug and serum-free culture medium can induce nucleus pulposus stem cells to enter the G0 phase.

[0094] Example 7

[0095] This example examines the effect of prepared nanopeptide drugs on the aging of nucleus pulposus stem cells (NPSCs):

[0096] G0 phase cells include three possibilities: terminally differentiated cells such as neurons or cardiomyocytes, senescent cells, and resting cells. Therefore, it is necessary to test the effect of prepared nanopeptide drugs on the aging of nucleus pulposus stem cells to determine their ability to induce resting phase. The method is as follows: Nucleus pulposus stem cells were divided into three groups: a conventional culture medium group (incubated in DMEM medium containing 10% fetal bovine serum), a prepared nanopeptide drug self-assembled particle suspension group (self-assembled particles + incubated in DMEM medium containing 10% fetal bovine serum, with a final concentration of 10 μg / mL), and a serum-free culture medium group (incubated in DMEM medium without fetal bovine serum). Each group was incubated in a cell culture incubator at 37℃ for 48 h. After removing the culture medium, the cells were washed three times with PBS. After washing with PBS, SA-β-Gal staining fixative (SA-β-Gal staining kit, Solarbio Life Science, China) was added and incubated for 15 min. After removing the fixative, the cells were washed three times with PBS and then SA-β-Gal working solution (SA-β-Gal staining kit, Solarbio Life Science, China) was added. The samples were incubated overnight at 37°C. The results were observed under white light using an inverted fluorescence microscope (Olympus IX73, Japan).

[0097] The results are as follows Figure 8 As shown in the figure (scale bar: 200μm), SA-β-Gal positive cells are senescent cells, and their quantitative results are as follows: Figure 9 As shown, the results indicate that incubation with the prepared nanopeptide drug and serum-free culture medium does not induce aging of nucleus pulposus stem cells. Therefore, combined with the results of Example 6, the prepared nanopeptide drug can induce nucleus pulposus stem cells to enter a quiescent state.

[0098] Example 8

[0099] Animal experiments:

[0100] (1) Grouping situation:

[0101] Male SD rats (6-8 weeks old) were randomly divided into 4 groups (n=9 per group). Each group was housed in a non-interventional environment for one week to allow for environmental acclimatization. The uninterventional rats served as the blank control group. Rats injected with culture medium into their caudal intervertebral discs were designated as the sham-operated group (sham-operated groups induce intervertebral disc degeneration), rats injected with nucleus pulposus stem cells into their caudal intervertebral discs were designated as the NPSCs group, and rats injected with extracellular matrix-cell complexes into their caudal intervertebral discs were designated as the complex group.

[0102] (2) Sample preparation and administration method:

[0103] Each rat in the sham-operated group was injected with 5 μL of culture medium; each rat in the NPSCs group was injected with 1.5 × 10⁻⁶ nucleus pulposus stem cells (NPSCs, P5). 4 / intervertebral disc, 5 μL / intervertebral disc; complex group: each rat injected with 1.5 × 10-1 nucleus pulposus stem cells (based on the amount of nucleus pulposus stem cells). 4 The intervertebral disc injection was 5 μL / disc. An extracellular matrix-cell complex (ECLC) complex needed to be prepared first. Specifically, a nanoparticle suspension with a concentration of 10 μg / mL was prepared according to the method in Example 2, and then incubated with nucleus pulposus stem cells (NPSCs, P5) at 37°C for 1 hour. Rats in each group were sacrificed by cervical dislocation at 3, 4, and 5 weeks after intervertebral disc injection, and data were collected.

[0104] (3) Immunofluorescence of tissue sections:

[0105] Intervertebral disc sections were examined 3 weeks post-surgery in both the NPSCs and complex groups. THT (thioflavin T) (green fluorescence) stained the β-sheet structure. Therefore, the sections were sequentially dewaxed, antigen retrieval was performed, blocked, incubated with CXCR1 immunofluorescence antibody, incubated with ThT, mounted with DAPI mounting solution, and observed under a laser confocal microscope (Zeiss 880, Germany).

[0106] The results are as follows Figure 10 As shown (scale bar: 10 μm), the quantitative results are as follows: Figure 11 As shown in the figure. The results indicate that after the extracellular matrix-cell complex is injected into the body, the nanopeptide drug self-assembly particles target the CXCR1 receptor, blocking the receptor and reducing the CXCR1 fluorescence signal, while the nanofibers with β-sheet structures (ThT signal) are significantly increased.

[0107] Immunofluorescence assays were performed on intervertebral disc sections from the complex group at 3, 4, and 5 weeks post-surgery, with the following results: Figure 12 As shown (scale bar: 10 μm), the quantitative results are as follows: Figure 13 The results showed that the β-sheet structure formed by the self-assembled nanopeptide drug particles did not change significantly, and the fluorescence of the CXCR1 receptor also showed no significant difference. Therefore, after binding with nucleus pulposus stem cells, the self-assembled nanopeptide drug particles can adhere to the surface of nucleus pulposus stem cells for a long time in vivo, providing support for exerting biological effects.

[0108] (4) Transmission electron microscopy observation of nucleus pulposus tissue:

[0109] The nucleus pulposus tissue of the blank control group and the complex group was observed by transmission electron microscopy at 3, 4, and 5 weeks postoperatively. Nucleus pulposus tissue was taken, fixed, stained, and then placed in a copper mesh for observation under a transmission electron microscope. Results are as follows: Figure 14 As shown (scale bar: 10 μm; magnification: 500 nm), the results indicate that the nanofiber structure (red arrow) formed by the deformation of the self-assembled nanopeptide drug particles can adhere to the surface of nucleus pulposus stem cells for a long time.

[0110] (5) H&E and safranin-fast green stained sections detection:

[0111] The blank control group, sham surgery group, NPSCs (P5) group, and complex group underwent coccygeal intervertebral disc sections at 3, 4, and 5 weeks post-operation. The sections were sequentially decalcified, embedded, sectioned, dewaxed, and stained with H&E or Safranin-Fix-Green before being observed under a light microscope. Results are as follows: Figure 15 As shown (scale bar: 100μm; magnified view: 20μm, red arrows / dashed lines: nucleus pulposus; blue arrows / dashed lines: nucleus pulposus stem cell clusters; green arrows / dashed lines: annulus fibrosus), histological scoring is as follows: Figure 16 As shown, the results indicate that NPSCs (P5) (senescent cells) and intervertebral disc puncture (sham surgery group) cause degeneration, which worsens over time. The extracellular matrix-cell complex involved in this invention inhibits the histological degeneration caused by both. Safranin-Fixed Green staining and its histological score ( Figure 17 (Scale bar: 500μm; Enlarged view: 100μm) and Figure 18 The results also show similar findings.

[0112] (6) MRI (Small Animal Magnetic Resonance Imaging) Examination:

[0113] The T2-weighted signal intensity of the caudal intervertebral discs in rats at 3, 4, and 5 weeks post-operation was measured using a 7.0T MRI system (MR Solutions, UK). The nucleus pulposus area at data acquisition time points was measured using ImageJ software. All images were measured blinded by two spinal surgeons.

[0114] The results are as follows Figure 19 As shown (scale bar: 3mm, yellow dashed circles indicate nucleus pulposus water content signal), the Pfirrmann score and quantitative results of nucleus pulposus area are as follows: Figure 20 and 21 As shown, the results indicate that NPSCs (P5) (senescent cells) significantly exacerbate nucleus pulposus degeneration, while the extracellular matrix-cell complex involved in this invention inhibits the progression of this degeneration. Disc puncture (sham surgery group) and injection of NPSCs (P5) (senescent cells) exacerbate nucleus pulposus degeneration, leading to a reduction in nucleus pulposus area. The extracellular matrix-cell complex involved in this invention inhibits the progression of nucleus pulposus area reduction and suppresses degeneration.

[0115] (7) p-mTOR immunofluorescence assay:

[0116] Tissue sections of rat caudal intervertebral discs were performed 3 weeks after surgery in the NPSCs (P5) group and the complex group. The method was as follows: after dewaxing, antigen retrieval with antigen extraction solution, heating, blocking for 40 minutes, and incubation with primary antibody (anti-p-mTOR (Ser2448)), followed by incubation with dye-labeled secondary antibody (AF-555 conjugated with goat anti-rabbit / rat IgG (H+L)). Finally, the results were observed by laser confocal microscope (Zeiss 880, Germany).

[0117] Staining results as follows Figure 22 As shown (scale bar: 100 μm; magnified view: 20 μm), the quantitative results of fluorescence intensity are as follows: Figure 23 and Figure 24 As shown. The results indicate that the extracellular matrix-cell complex involved in this invention inhibits the expression of p-mTOR in vivo, and its inhibition of intervertebral disc degeneration may be achieved by inhibiting the PI3K / Akt / mTOR pathway.

[0118] The applicant declares that this invention illustrates an extracellular matrix-cell complex for inhibiting intervertebral disc degeneration, its preparation method, and its application through the above embodiments. However, this invention is not limited to the above embodiments, meaning that this invention does not necessarily rely on the above embodiments for implementation. Those skilled in the art should understand that any improvements to this invention, equivalent substitutions of raw materials, additions of auxiliary components, and selection of specific methods, etc., all fall within the protection and disclosure scope of this invention.

[0119] The preferred embodiments of the present invention have been described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solution of the present invention, and these simple modifications all fall within the protection scope of the present invention.

[0120] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present invention will not describe the various possible combinations separately.

Claims

1. An extracellular matrix-cell complex for inhibiting degeneration of an intervertebral disc, characterized in that, The extracellular matrix-cell complex that inhibits intervertebral disc degeneration includes modified cells and biomimetic extracellular matrix nanofibers constructed on the surface of the modified cells. The modified cells were selected from nucleus pulposus stem cells; The biomimetic extracellular matrix nanofibers are obtained by the self-assembly of nanopeptide drugs and their interaction with the modified cells through ligand-receptor interactions, resulting in deformation. The nanopeptide drug comprises a hydrophobic unit, a self-assembly unit, and a targeting unit connected in sequence; the hydrophobic unit is linoleic acid; the self-assembly unit is a polypeptide sequence of FFVLK; and the targeting unit is a CXCR1 ligand with the sequence FKPHFPKSYTKICQ.

2. The method of claim 1, wherein the extracellular matrix-cell complex is prepared by the steps of: a) providing a population of cells; b) providing a matrix; c) mixing the population of cells and the matrix; and d) incubating the mixture of step c) for a period of time sufficient to allow the cells to attach to the matrix. The preparation method includes: Nanopeptide drugs are self-assembled to form nanoparticles, which are then mixed with modified cells and co-incubated to obtain the extracellular matrix-cell complex that inhibits intervertebral disc degeneration.

3. The method of claim 2, wherein the extracellular matrix-cell complex is prepared by the steps of: a) providing a population of cells; b) providing a matrix; c) combining the population of cells and the matrix; and d) incubating the combined population of cells and matrix in a medium suitable for cell growth. In the co-incubation system, the concentration of the nanoparticle is 5-100 μg / mL; the concentration of the modified cell is 2 x 10 6 -4 x 10 6 / mL.

4. The method of claim 3, wherein the extracellular matrix-cell complex is prepared by the steps of: a) providing a population of cells; b) providing a matrix; c) mixing the population of cells and the matrix; and d) incubating the mixture of step c) for a period of time sufficient to allow the cells to attach to the matrix. In the co-incubation system, the concentration of the nanoparticle is 5-20 μg / mL; the concentration of the modified cell is 2 x 10 6 -4 x 10 6 / mL.

5. The method of claim 2, wherein the extracellular matrix-cell complex is prepared by the steps of: a) providing a population of cells; b) providing a matrix; c) combining the population of cells and the matrix; and d) culturing the cells in the matrix. The nanopeptide drug was prepared by solid-phase synthesis.

6. The method of claim 2, wherein the extracellular matrix-cell complex is prepared by the steps of: a) providing a population of cells; b) providing a matrix; c) combining the population of cells and the matrix; and d) culturing the cells in the matrix. The co-incubation was carried out at 35-39℃ for 1-4 hours.

7. The application of the extracellular matrix-cell complex for inhibiting intervertebral disc degeneration as described in claim 1 in the preparation of intervertebral disc implant materials with the function of inhibiting intervertebral disc degeneration.

8. The use of the extracellular matrix-cell complex for inhibiting intervertebral disc degeneration according to claim 1 in the preparation of a pharmaceutical formulation for treating intervertebral disc degeneration.

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