Self-assembled stem cell material composite spheroids, and methods of making and using the same

Abstract

The present application relates to the technical field of regenerative medicine, in particular to a self-assembled stem cell material composite spheroid and a preparation method and application thereof.The preparation method comprises the following steps: preparing a primary mesenchymal stem cell suspension; taking animal tissue and preparing a decellularized matrix microparticle suspension through decellularization treatment; mixing a nanosheet solution and a TGF-beta solution and incubating to obtain a factor-loaded nanosheet solution; uniformly mixing the factor-loaded nanosheet solution and the decellularized matrix microparticle suspension, cross-linking and compounding to obtain cross-linked composite microparticles; and mixing the cross-linked composite microparticle suspension and the stem cell suspension and culturing to obtain a self-assembled stem cell material composite spheroid.The self-assembled stem cell material composite spheroid has a high survival rate when implanted into an intervertebral disc, controllable differentiation, and a good clinical application prospect, and provides a new idea for the treatment of degenerative intervertebral discs and other degenerative diseases.

Description

Technical Field

[0001] This invention relates to the field of regenerative medicine technology, and more specifically to a self-assembled stem cell material composite sphere, its preparation method, and its application. Background Technology

[0002] Low back pain is a very common musculoskeletal disorder and a leading cause of disability. Intervertebral disc degeneration is widely recognized as a major contributing factor to low back pain and is closely related to its severity. Current clinical treatments for intervertebral disc degeneration include conservative and surgical therapies. However, these treatments only alleviate the clinical symptoms caused by the herniation and cannot reverse the degeneration; patients still face the risk of recurrence. With the development of tissue engineering technology, attention has increasingly focused on stem cell-based tissue engineering therapies. Because the intervertebral disc is the largest avascular closed tissue in the human body, characterized by low oxygen levels, low cell count, and poor self-repair capabilities, local transplantation of exogenous stem cells holds great potential for restoring disc structure and function. However, stem cell therapy for intervertebral disc degeneration is limited by low cell survival rates due to the degenerated microenvironment and the difficulty in controlling the directed differentiation of stem cells. Therefore, using tissue engineering methods combined with biomaterials and growth factors to reshape the microenvironment for stem cell survival is of significant research and clinical importance for fully leveraging the therapeutic effects of stem cells and achieving regeneration and repair of intervertebral discs or other damaged tissues.

[0003] In recent years, stem cell therapy has focused primarily on simple two-dimensional stem cell injections, or on leveraging various tissue engineering methods based on or without biomaterial scaffolds to advance stem cell therapy. While some progress has been made, achieving regeneration and repair of degenerated intervertebral discs still faces several obstacles and challenges, such as the low retention rate of two-dimensional stem cells after injection. In tissue engineering methods based on biomaterial scaffolds, natural or synthetic hydrogel materials are widely used for cell loading due to their tunable mechanical properties, injectability, and good cell compatibility. However, hydrogel-based stem cell culture may hinder intercellular interactions and three-dimensional cell aggregation, negatively impacting the regeneration and repair of the nucleus pulposus and cartilage, or leading to toxic side effects from scaffold degradation. Scaffold-free tissue engineering techniques, such as three-dimensional stem cell spheres and two-dimensional stem cell sheets assembled from stem cells, effectively preserve intercellular interactions during tissue regeneration and avoid the side effects of in vivo and exogenous scaffold materials, and have gained increasing attention in recent years. However, these traditional stem cell assembly methods still present some problems with three-dimensional stem cell spheres, including limited nutrient diffusion, core necrosis, uncontrollable stem cell differentiation, and a low ability to regulate inflammation and inhibitory microenvironments at the site of tissue damage.

[0004] Therefore, existing stem cell therapy programs all have obvious shortcomings. It is essential to develop a new method that can fully realize the therapeutic potential of stem cells, achieve effective microenvironment regulation, and prepare stem cells with high survival rate and efficient directed differentiation in vivo. Summary of the Invention

[0005] To address the shortcomings of the above-mentioned technical solutions, the present invention aims to provide a method for preparing self-assembled stem cell material composite spheres.

[0006] Another object of the present invention is to provide a self-assembled stem cell material composite sphere obtained by the above preparation method.

[0007] Another object of the present invention is to provide the application of the above-mentioned self-assembled stem cell material composite spheres.

[0008] The objective of this invention is achieved through the following technical solution.

[0009] This invention provides a method for preparing self-assembled stem cell material composite spheres, comprising the following steps:

[0010] Step 1: Primary mesenchymal stem cells were isolated using the tissue adherence method, cultured in complete culture medium, and digested to obtain a stem cell suspension;

[0011] Step 2: After disinfecting and sterilizing animal tissue, decellularize it to obtain decellularized matrix (ECM), then freeze-dry, grind, resuspend, and filter to obtain a decellularized matrix microparticle suspension.

[0012] Step 3: Mix and incubate the nanosheet solution with the TGF-β solution to load TGF-β onto the nanosheets, thereby obtaining a nanosheet loading factor solution. The concentration of the nanosheet solution is 0.1–5 mg / ml, the concentration of the TGF-β solution is 10–500 ng / ml, and the mixing ratio of the nanosheet solution to the TGF-β solution is (0.1–5) ml: (10–500) ng.

[0013] Step 4: Mix the factor-carrying nanosheet solution obtained in Step 3 and the decellularized matrix microparticle suspension obtained in Step 2 evenly, crosslink and incubate at 4°C for 12-48 h to obtain crosslinked composite microparticles. The concentration of the factor-carrying nanosheet solution is 0.1-1 mg / ml, the concentration of the decellularized matrix microparticle suspension is 1-50 mg / ml, and the volume ratio of the factor-carrying nanosheet solution to the decellularized matrix microparticle suspension is (1-10):(1-10).

[0014] Step 5: Mix the suspension of cross-linked composite microparticles obtained in Step 4 with the stem cell suspension obtained in Step 1, seed the mixture in well plates, centrifuge to promote cell aggregation at the bottom of the wells, and culture to obtain self-assembled stem cell material composite spheroids. The concentration of the cross-linked composite microparticle suspension is 0.1–5 mg / ml, and the concentration of the stem cell suspension is 1–10 × 10⁻⁶ mg / ml. 5 The volume ratio of cross-linked composite microparticle suspension to stem cell suspension is (1-2):(1-10).

[0015] In the above technical solution, further, in step 1, the primary mesenchymal stem cells are primary bone marrow mesenchymal stem cells, primary adipose mesenchymal stem cells, or primary umbilical cord mesenchymal stem cells.

[0016] In the above technical solution, further, in step 1, the culture is carried out in an incubator, and the complete stem cell culture medium needs to be replaced every 3 days until the cell confluence is greater than 90%. The complete stem cell culture medium is DMEM / F12 culture medium containing 10% fetal bovine serum and 1% antibiotics.

[0017] In the above technical solution, further, in step 1, the primary mesenchymal stem cells are cultured in a cell culture incubator to the 2nd to 5th generation.

[0018] In the above technical solution, further, in step 2, the animal tissue is nucleus pulposus tissue, cartilage tissue, muscle tissue, heart, liver, or kidney.

[0019] In the above technical solution, further, in step 2, the disinfection and sterilization involves immersing the animal tissue in 0.1% peracetic acid for 4 hours and then rinsing it with sterile water or physiological saline.

[0020] In the above technical solution, further, in step 2, the decellularization process involves decellularizing the sterilized animal tissue with 1% SDS and 1% Triton X-100 for 3 days, washing it with a buffer containing 50 U / mL DNase I and 1 U / mL RNase, and then washing it with sterile water to obtain a decellularized matrix.

[0021] In the above technical solution, further, in step 2, the freeze-drying grinding is performed by quick freezing with liquid nitrogen, followed by thorough grinding 5 to 10 times in a grinder, sterilization with ultraviolet light for 1 hour, resuspending in DMEM / F12 basal culture medium, mixing by pipetting, filtering, and obtaining a decellularized matrix microparticle suspension with a particle size of 20 to 100 μm.

[0022] In the above technical solution, further, in step 3, the mixing and incubation involves mixing the nanosheet solution and the TGF-β solution, incubating at 4°C for 12-48 hours, and centrifuging to obtain the carrier factor nanosheet solution.

[0023] In the above technical solution, further, in step 3, the nanosheets are titanium carbide nanosheets, manganese dioxide nanosheets, graphene nanosheets, or black phosphorus nanosheets.

[0024] The above technical solution further includes preparing a titanium carbide nanosheet solution using an etching and exfoliation method, comprising the following steps: Ti3AlC2 powder is added to an HF solution and reacted for 5 hours under stirring. The mixture is then centrifuged and repeatedly washed until the pH reaches 4-5, yielding a black precipitate. TPAOH is added and mixed thoroughly. The mixture is then centrifuged and washed multiple times until the pH reaches 7. Distilled water is added, followed by sonication, centrifugation, and dilution to obtain the titanium carbide nanosheet solution. The mass fraction of Ti3AlC2 is 1, the volume fraction of HF is 20, and the volume fraction of TPAOH is 20. The volume fractions are measured in ml, and the mass fractions are measured in g.

[0025] In the above technical solution, the dilution is further described as diluting with deionized water to 500 μg / ml.

[0026] In the above technical solution, the repeated washing is performed with distilled water and ethanol, the mixing time is 24 hours, the mixing temperature is room temperature, the centrifugation rate is 5000 rpm, and the ultrasonication time is 2 hours.

[0027] In the above technical solution, further, in step 5, the well plate is a low-adhesion U-shaped 96-well plate, the centrifugation rate is 1000 rpm, the time is 3 min, the incubation temperature is 37℃, and the incubation time is 12-72 h.

[0028] The present invention also provides a self-assembled stem cell material composite sphere, which is prepared by the above preparation method.

[0029] Furthermore, in the above-mentioned technical solution, the application of the self-assembled stem cell material composite spheres in the treatment of intervertebral disc degeneration involves in-situ injection of the self-assembled stem cell material composite spheres to replenish nucleus pulposus cells.

[0030] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0031] 1. The self-assembled stem cell material composite spheres of the present invention are incorporating decellularized matrix microparticles of the nucleus pulposus, thereby providing a pseudo-natural microenvironment for the self-assembled stem cell material composite spheres and promoting their proliferation and chondrogenic differentiation. At the same time, the present invention uses titanium carbide nanosheets to scavenge reactive oxygen species (ROS) and loads transforming growth factor-β (TGF-β) on the titanium carbide nanosheets, which together create a physical microenvironment for the growth and differentiation of the self-assembled stem cell material composite spheres, thereby improving the oxidative microenvironment of degenerated intervertebral discs.

[0032] 2. The intervertebral disc is the largest avascular tissue in the human body, with a low cell count and poor self-repair ability; it cannot achieve tissue repair solely through the regeneration of its own endogenous cells. Therefore, this invention exposes the rat intervertebral disc and injects self-assembled stem cell material composite spherical bodies to supplement nucleus pulposus cells, thereby improving the intervertebral disc's repair capacity. The self-assembled stem cell material composite spherical bodies of this invention exhibit high survival rates and controllable differentiation when implanted into the intervertebral disc. This invention's self-assembled stem cell material composite spherical bodies have promising clinical application prospects and provide new ideas for the treatment of degenerated intervertebral discs and other degenerative diseases.

[0033] 3. The decellularized nucleus pulposus matrix of this invention is derived from the extracellular matrix of natural tissue (nucleus pulposus tissue from fresh bovine vertebrae). It retains some protein components and biochemical clues of natural tissue, making it well-suited for reconstructing the stem cell microenvironment. The titanium carbide nanosheets of this invention have excellent ROS scavenging and anti-inflammatory effects, effectively improving the oxidative stress microenvironment of degenerated intervertebral discs. In addition, the high surface area of ​​titanium carbide nanosheets allows for efficient loading of TGF-β, which is continuously released as it degrades, making it an excellent drug delivery carrier. The titanium carbide nanosheets and decellularized nucleus pulposus matrix microparticles are cross-linked, which on the one hand prevents endocytosis, and on the other hand improves the overall mechanical strength of the cross-linked composite microparticles, slowing down the degradation rate of ECM microparticles. The two work together to create conditions for the survival and differentiation of self-assembled stem cell material composite spherical bodies, thereby promoting the regeneration and repair of degenerated intervertebral discs. Attached Figure Description

[0034] Figure 1 Images of the natural nucleus pulposus and its decellularized matrix, along with DAPI staining results;

[0035] Figure 2 Determination of DNA content in natural nucleus pulposus and decellularized matrix of nucleus pulposus;

[0036] Figure 3 The difference lies in the protein composition between the natural nucleus pulposus and the decellularized matrix of the nucleus pulposus;

[0037] Figure 4 For the biocompatibility of decellularized matrix microparticles in the nucleus pulposus;

[0038] Figure 5 TEM image of titanium carbide nanosheets;

[0039] Figure 6 The Zeta potential diagram of titanium carbide nanosheets;

[0040] Figure 7 This is a particle size distribution diagram of titanium carbide nanosheets;

[0041] Figure 8 This is a graph showing the biocompatibility and concentration screening of titanium carbide nanosheets.

[0042] Figure 9 This is a diagram showing the antioxidant activity of titanium carbide nanosheets;

[0043] Figure 10 TEM images of self-assembled stem cell material composite spheres and stem cell spheres;

[0044] Figure 11 The expression of stem cell-like chondrogenic differentiation markers in composite spheres made of different materials;

[0045] Figure 12 This describes the survival and differentiation of the composite spherical bodies after implantation. Detailed Implementation

[0046] The technical solution of the present invention will be further described below with reference to specific embodiments.

[0047] Example 1

[0048] A method for preparing self-assembled stem cell material composite spheres includes the following steps:

[0049] Step 1: Primary bone marrow mesenchymal stem cells were isolated using the tissue adherence method and cultured in a cell culture incubator in complete culture medium to obtain a concentration of 4 × 10⁻⁶ cells / mL. 5 The stem cell suspension is prepared in cells per ml. The complete stem cell culture medium in the cell culture incubator needs to be changed every 3 days. The cells are cultured to the 3rd generation until the confluence of cells is greater than 90%. The complete stem cell culture medium is DMEM / F12 containing 10% fetal bovine serum (FBS) and 1% antibiotics (PS).

[0050] The specific process for isolating primary bone marrow mesenchymal stem cells using the tissue adherence method is as follows: Rats are euthanized by dislocation, soaked in 75% ethanol for 20 minutes, and the bilateral femurs and tibias are aseptically separated on a sterile operating table. DMEM / F12 basal medium is drawn from the rat bone marrow and flushed out of the medullary cavity of the tibia and femur using a 10 ml syringe. The cells are centrifuged at 1000 rpm for 5 minutes, the supernatant is discarded, and erythrocyte lysis buffer is added to the cell pellet to lyse the erythrocytes. The remaining bone marrow cells are centrifuged again and resuspended in DMEM / F12 complete medium supplemented with 10% FBS and 1% PS (penicillin and streptomycin). The cells are then cultured routinely in a CO2 incubator. After cell adhesion, the cells are passaged using trypsin digestion, and the third-generation bone marrow mesenchymal stem cells are used for subsequent experiments.

[0051] Step 2: Nucleus pulposus tissue is taken from fresh bovine vertebrae, sterilized, and then decellularized to obtain decellularized nucleus pulposus matrix. This matrix is ​​then freeze-dried, ground, sterilized, resuspended, and filtered to obtain a microparticle suspension of decellularized nucleus pulposus matrix. The sterilization process involves disinfection with 0.1% peracetic acid solution for 4 hours, followed by washing with sterile water. The decellularization process involves sterile 1% (w / v) SDS solution and 1% Triton [agent]. X-100 solution was used to remove cells by shaking on a shaker at room temperature, with the medium changed every 4 hours for three consecutive days. The solution was then washed with sterile water until no foam was present. The solution was washed at 37°C with Tris-HCl buffer containing 50 U / mL DNase I and 1 U / mL ribonuclease A, followed by washing with sterile water. The lyophilization and grinding process involved rapid freezing in liquid nitrogen, followed by placement in grinding tubes and low-temperature grinding 5–10 times in a grinder. The mixture was then sterilized with UV light for 1 hour, resuspended in DMEM / F12 basal medium, thoroughly mixed by pipetting, and filtered through a 70 μm pore size filter to obtain a suspension of decellularized nucleus pulposus matrix microparticles (particle size less than 70 μm).

[0052] Step 3: The titanium carbide nanosheet solution and the TGF-β solution are mixed and incubated to load TGF-β onto the titanium carbide nanosheets, resulting in a loading factor nanosheet solution. The concentration of the titanium carbide nanosheet solution is 800 μg / ml, and the concentration of the TGF-β solution is 500 ng / ml. The mixing ratio of the nanosheet solution to the TGF-β solution is 1 ml: 500 ng. The mixing and incubation are performed by mixing the titanium carbide nanosheet solution and the TGF-β solution, incubating at 4°C for 24 h, centrifuging at 12000 rpm for 10 min, discarding the supernatant, and resuspending the precipitate in distilled water to obtain the loading factor nanosheet solution.

[0053] Titanium carbide nanosheet solution was prepared by etching and exfoliation method, including the following steps: 1g Ti3AlC2 powder was added to 20ml HF solution (40wt.%) under magnetic stirring and reacted for 5 hours. After centrifugation, the solution was repeatedly washed with distilled water and anhydrous ethanol until the pH reached 4-5, resulting in a black precipitate. 20ml TPAOH (25wt.%) was added at room temperature and mixed for 24 hours until homogeneous. The solution was then washed by centrifugation at 5000rpm multiple times until the pH reached 7. Distilled water was added, and the solution was sonicated for 2 hours, followed by centrifugation at 3500rpm for 1 hour. The solution was diluted with deionized water to 500μg / ml to obtain titanium carbide nanosheet solution.

[0054] Step 4: Mix the titanium carbide factor-carrying nanosheet solution and the decellularized nucleus pulposus matrix microparticle suspension evenly, and incubate at 4°C for 24 h to achieve cross-linking and composite, obtaining cross-linked composite microparticles. The concentration of the factor-carrying nanosheet solution is 0.5 mg / ml, the concentration of the decellularized nucleus pulposus matrix microparticle suspension is 10 mg / ml, and the volume ratio of the titanium carbide factor-carrying nanosheet solution to the decellularized nucleus pulposus matrix microparticle suspension is 1:2.5. Step 5: Mix 20 μl of a 5 mg / ml suspension of cross-linked composite microparticles with 20 μl of a 4 × 10⁻⁶ m³ / ml suspension of cross-linked composite microparticles. 5 The stem cell suspension was mixed with cells / ml and seeded into a low-adhesion U-shaped 96-well plate. 150 μl of DMEM / F12 complete medium was added, and the mixture was centrifuged at 1000 rpm for 3 min to promote cell aggregation at the bottom of the well. The mixture was then cultured in an incubator at 37°C for 24 h to obtain self-assembled stem cell material composite spheres.

[0055] Comparative Example 1

[0056] At a concentration of 20 μl, the concentration is 4 × 10⁻⁶. 5 The third-generation bone marrow mesenchymal stem cell suspension obtained in step 1 of Example 1 was seeded into a low-adhesion U-shaped 96-well plate, supplemented with 150 μl of DMEM / F12 complete culture medium, centrifuged at 1000 rpm for 3 min to promote cell aggregation at the bottom of the well, and cultured in an incubator at 37°C for 24 h to obtain stem cell spheroids. The bone marrow mesenchymal stem cells used were the third-generation bone marrow mesenchymal stem cells described in the example.

[0057] The decellularized nucleus pulposus matrix obtained in Example 1 was characterized for decellularization: a small piece of the decellularized nucleus pulposus matrix was fixed in 4% paraformaldehyde for 5 hours to obtain fixed tissue. The fixed tissue was then dehydrated with 30% sucrose solution for 24 hours, embedded in OCT, frozen, and cut into 8 μm slices for storage at -20°C. DAPI staining was used to examine the removal of cell nuclei in the nucleus pulposus tissue before (nucleus pulposus tissue) and after (decellularized nucleus pulposus matrix) decellularization treatment. The DNA content in the natural nucleus pulposus tissue and the decellularized nucleus pulposus matrix was quantitatively analyzed using a DNA quantification kit. Figure 1 and Figure 2 As shown.

[0058] Depend on Figure 1 and Figure 2 It can be seen that the nucleus pulposus tissue became fluffy and swollen after decellularization, the cell nuclei were basically removed, and the detected DNA content was less than 50 ng / mg, indicating that the decellularization method has a good decellularization effect and meets the application requirements of low immunogenicity.

[0059] The decellularized nucleus pulposus matrix obtained in Example 1 was subjected to proteomics identification. The main procedures included protein digestion, LC-MS / MS analysis, database retrieval, and data analysis. This test was completed and experimental results were obtained from Shanghai Baipu Biotechnology Co., Ltd., as shown in the attached report. Figure 3 As shown.

[0060] Depend on Figure 3 It is known that the number of protein types in the decellularized matrix of the nucleus pulposus is slightly lower than that in the natural nucleus pulposus tissue, and the common protein components account for the majority. This indicates that the decellularization treatment of the present invention can well preserve the protein components in the original nucleus pulposus tissue, giving it good biological activity.

[0061] Cell compatibility assessment:

[0062] Third-generation bone marrow mesenchymal stem cells were used at a rate of 3 × 10⁻⁶. 4 Cells were seeded at a density of 1 cell / well in 96-well low-adhesion plates, and then different concentrations of decellularized nucleus pulposus matrix microparticle suspension (50, 100, 200, and 400 μg / ml) were added. The plates were then incubated at 37°C to form cell spheroids. After 24 hours, the plates were removed, and 10 μl of CCK8 working solution was added. The plates were incubated at 37°C for 2 hours, and the absorbance was measured using a microplate reader. The results are shown below. Figure 4 As shown. By Figure 4 It can be seen that adding different concentrations of decellularized nucleus pulposus microparticles to bone marrow mesenchymal stem cells not only did not produce cytotoxicity, but also showed better results than the control group ( Figure 4 Compared with the group with an ECM concentration of 0, all three groups significantly promoted the proliferation of bone marrow mesenchymal stem cells, especially at a concentration of 100 μg / ml, which showed the most significant promoting effect. This indicates that the decellularized nucleus pulposus matrix microparticles have excellent cell compatibility. The above proteomics identification results also explain the source of the bioactivity of the decellularized nucleus pulposus matrix microparticles.

[0063] Microstructure of titanium carbide nanosheets:

[0064] A solution of titanium carbide nanosheets was dropped onto a copper mesh sample stage and dried. The drying time was approximately one hour. After drying, the nanosheets were observed and photographed using a transmission electron microscope (TEM). Figure 5As shown, by Figure 5 It can be seen that the present invention successfully prepared ultrathin monolayer sheet-like titanium carbide nanosheets using an etching and exfoliation method.

[0065] Potential and particle size of titanium carbide nanosheets:

[0066] Titanium carbide nanosheet solution was diluted with ultrapure water, then poured into a cuvette, and its potential and particle size were measured using a nanolaser particle size analyzer. The results are as follows: Figure 6 and Figure 7 As shown, by Figure 6 and Figure 7 It can be seen that titanium carbide nanosheets carry a negative charge with a potential of about 20mV, and their particle size is mainly distributed in the range of 100-400nm, indicating that titanium carbide nanosheets have been successfully prepared.

[0067] Cellular compatibility of titanium carbide nanosheets:

[0068] Bone marrow mesenchymal stem cells were used at a rate of 3 × 10⁻⁶. 4 Titanium carbide nanosheets were seeded at a density of 10, 20, 40, 80, and 160 μg / ml in 96-well low-adhesion plates. Different concentrations of titanium carbide nanosheet solution (10, 20, 40, 80, and 160 μg / ml) were added to the culture medium, and the plates were incubated at 37°C to form cell spheroids. On the first and second days after spheroidization, the plates were removed at the same time point, and 10 μl of CCK8 working solution was added. The plates were incubated at 37°C for 2 hours, and the absorbance was measured using a microplate reader. The results are as follows: Figure 8 As shown, by Figure 8 It can be seen that the addition of titanium carbide nanosheets at different concentrations had different effects on the activity of bone marrow mesenchymal stem cells. High concentrations of titanium carbide nanosheets added to bone marrow mesenchymal stem cells showed obvious cytotoxicity with the extension of culture time, while low concentrations of titanium carbide nanosheets had little effect on the activity of bone marrow mesenchymal stem cells.

[0069] Antioxidant activity of titanium carbide nanosheets:

[0070] The ability of titanium carbide nanosheets to scavenge reactive oxygen species (ROS) was assessed using bone marrow mesenchymal stem cells (BMSCs) in an oxidative microenvironment. Specifically, BMSCs were prepared at 3 × 10⁶ cells per well. 4 Cells were seeded in low-adhesion U-shaped 96-well plates and co-incubated with titanium carbide nanosheets of different concentrations (20, 40 μg / mL) to form composite cell spheroids. After spheroid formation, the cells were treated with 100 μM H₂O₂ for 2 h, followed by the addition of 10 μL of CCK8 working solution and incubation at 37°C for 2 h. The absorbance was measured using a microplate reader. The results are shown below. Figure 9 As shown, by Figure 9It can be seen that H2O2 stimulation significantly increases the mortality rate of bone marrow mesenchymal stem cells, while the addition of titanium carbide nanosheets of different concentrations significantly improves the survival rate of bone marrow mesenchymal stem cells. This indicates that titanium carbide nanosheets have good antioxidant properties, play an effective protective role, and promote the survival of bone marrow mesenchymal stem cells.

[0071] The self-assembled stem cell material composite spheroids prepared in Example 1 and the stem cell spheroids prepared in Comparative Example 1 were respectively fixed in 2.5% v / v glutaraldehyde solution, followed by gradient dehydration with 50%, 70%, 100%, and 100% ethanol for 10 minutes at each concentration. After dehydration, the self-assembled stem cell material composite spheroids and stem cell spheroids were removed from 100% ethanol and placed in a supercritical dryer for critical point drying. The drying time was approximately 1 hour. After drying, they were removed, fixed on a sample stage, sputtered with gold, and their microstructure was observed using a scanning electron microscope (SEM) at an accelerating voltage of 5 kV. The results are as follows: Figure 10 As shown. By Figure 10 It was observed that both the self-assembled stem cell material composite spheroids and the stem cell spheroids were dense and compact. The stem cell spheroids showed obvious cell clusters and less extracellular matrix secretion, while the self-assembled stem cell material composite spheroids secreted a large amount of extracellular matrix, integrating the decellularized nucleus pulposus matrix and titanium carbide nanosheets with the cells without clear boundaries, demonstrating better integration. This indicates that the addition of decellularized bone marrow matrix and titanium carbide nanosheets to the self-assembled stem cell material composite spheroids improved the overall vitality of stem cells and facilitated the provision of a favorable microenvironment for stem cells.

[0072] Evaluation of the stem cell differentiation-promoting effect of the self-assembled stem cell material composite spheres prepared in this invention:

[0073] We used quantitative real-time PCR to detect the expression of chondrogenic marker genes in different cell spheroid groups: pure stem cell spheroids (BMSCs), titanium carbide nanosheet composite stem cell spheroids (NSs+BMSCs), TGF-β factor composite stem cell spheroids (TGF+BMSCs), factor-carrying nanosheet composite stem cell spheroids (NSs-TGF+BMSCs), matrix microparticle composite stem cell spheroids (ECM+BMSCs), and factor-carrying nanosheet matrix microparticle composite stem cell spheroids (ECM-NSs-TGF+BMSCs). Each group was prepared by using bone marrow mesenchymal stem cells at a density of 3 × 10⁶ cells per well, following the method described in Example 1. 4Cells were seeded in low-adhesion U-shaped 96-well plates at a specific ratio. The difference between each group was the addition of the aforementioned titanium carbide nanosheets (NSs+BMSC group), the aforementioned TGF-β factor (TGF+BMSC), the aforementioned factor-carrying nanosheets (NSs-TGF+BMSC), the aforementioned matrix microparticles (ECM+BMSC), and the aforementioned factor-carrying nanosheet matrix microparticles (ECM-NSs-TGF+BMSC) to obtain spheroids, which were then incubated at 37°C for 7 days. Specifically, the cell spheroids from each group were first collected, and 1 ml of TRIZOL was added and mixed thoroughly. The mixture was then transferred to EP tubes for RNA extraction according to the RNA extraction kit instructions. RNA concentration was then measured using Nanodrop. Next, RNA was reverse transcribed to obtain cDNA. Finally, primers and fluorescent dyes were added for detection. The results are shown below. Figure 11 As shown. By Figure 11 It is evident that both decellularized nucleus pulposus matrix microparticles and titanium carbide nanosheets, when added to cell spheres, can more or less enhance the expression of differentiation genes, and the combined addition of both and the loading of factors can synergistically promote stem cell differentiation. The expression trends of differentiation markers in the early, middle, and late stages are consistent. This confirms the differentiation-promoting effects of matrix microparticles and titanium carbide nanosheets.

[0074] Evaluation of implantation in vivo:

[0075] First, a rat caudal intervertebral disc degeneration model was constructed. Specifically, approximately 20 rats were randomly divided into 6 groups: normal control group, defect group, pure cell spheroid group, ECM+ cell spheroid group, ECM-NSs+ cell spheroid group, and ECM-NSs-TGF+ cell spheroid group, with n=6 intervertebral discs in each group. Rats were anesthetized by intraperitoneal injection of sodium pentobarbital. The skin of the rat tail was incised to expose the intervertebral discs. A sterile 20-gauge needle was inserted into the center of the nucleus pulposus to aspirate the nucleus pulposus tissue, inducing disc degeneration. Subsequently, equal amounts of GFP-fluorescent cell spheroids from each group were injected into each nucleus pulposus using a microinjector. The defect group was injected with PBS as a control. The outer skin was then sutured, disinfected with iodine, and the rats were fed routinely.

[0076] One week later, the intervertebral disc was removed and sectioned, and the GFP fluorescence signal in different groups was observed under a confocal microscope. Figure 12As can be seen, different cell sphere groups retained fluorescence signals to varying degrees after injection. With the addition of the composite components, the intracellular connections of the cell spheres gradually tightened, the post-injection loss rate gradually decreased, and the fluorescence signal gradually increased. The group containing factor-carrying titanium carbide nanosheets and decellularized nucleus pulposus matrix microparticles combined with stem cell spheres (ECM-NSs-TGF+ cell spheres) exhibited the highest fluorescence area retention. Furthermore, in the cell sphere group with ECM composite, not only was enhanced fluorescence signal observed, but importantly, some cell sphere residue was also visible, indicating that the composite spheres were not completely degraded one week after injection and remained in situ. This further confirms the local retention capacity of the composite spheres in the harsh intervertebral disc environment and demonstrates long-term therapeutic effects through an appropriate degradation rate.

[0077] The present invention has been described above by way of example. It should be noted that any simple modifications, alterations or other equivalent substitutions that can be made by those skilled in the art without creative effort without departing from the core of the present invention fall within the protection scope of the present invention.

Claims

1. A method for preparing self-assembled stem cell material composite spheres, characterized in that, Includes the following steps: Step 1: Prepare a suspension of primary mesenchymal stem cells; Step 2: Take animal tissue, decellularize it to obtain decellularized matrix, and then prepare a decellularized matrix microparticle suspension; Step 3: Mix and incubate the nanosheet solution with the TGF-β solution to obtain a carrier factor nanosheet solution, wherein the concentration of the nanosheet solution is 0.1~5 mg / ml, the concentration of the TGF-β solution is 10~500 ng / ml, and the mixing ratio of the nanosheet solution to the TGF-β solution is (0.1~5) ml: (10~500) ng; Step 4: Mix the factor-carrying nanosheet solution obtained in Step 3 and the decellularized matrix microparticle suspension obtained in Step 2 evenly, crosslink and composite them to obtain crosslinked composite microparticles. The concentration of the factor-carrying nanosheet solution is 0.1~1 mg / ml, the concentration of the decellularized matrix microparticle suspension is 1~50 mg / ml, and the volume ratio of the factor-carrying nanosheet solution to the decellularized matrix microparticle suspension is (1~10):(1~10). Step 5: Mix the suspension of cross-linked composite microparticles obtained in Step 4 with the stem cell suspension obtained in Step 1, and culture to obtain self-assembled stem cell material composite spheres. The concentration of the cross-linked composite microparticle suspension is 0.1–5 mg / ml, and the concentration of the stem cell suspension is 1–10 × 10⁻⁶ mg / ml. 5 The volume ratio of the cross-linked composite microparticle suspension to the stem cell suspension was (1~2): (1~10). The primary mesenchymal stem cells mentioned are primary bone marrow mesenchymal stem cells. In step 2, the animal tissue is nucleus pulposus tissue; The decellularization process involves decellularizing the sterilized animal tissue with 1% SDS and 1% Triton X-100, washing it with a buffer containing 50 U / mL DNase I and 1 U / mL RNase, and then washing it with sterile water to obtain a decellularized matrix; the particle size of the obtained decellularized matrix microparticle suspension is 20~100 μm. In step 3, the nanosheets are titanium carbide nanosheets, manganese dioxide nanosheets, graphene nanosheets, or black phosphorus nanosheets.

2. The preparation method according to claim 1, characterized in that, In step 1, primary mesenchymal stem cells are isolated using the tissue adherence method and cultured in complete culture medium until the cell confluence is greater than 90%; the complete stem cell culture medium is DMEM / F12 culture medium containing 10% fetal bovine serum and 1% antibiotics.

3. The preparation method according to claim 1, characterized in that, In step 1, the primary mesenchymal stem cells are cultured to the 2nd to 5th generation.

4. The preparation method according to claim 1, characterized in that, In step 3, the mixing and incubation involves mixing the nanosheet solution and the TGF-β solution, incubating at 4°C for 12-48 hours, and centrifuging to obtain the carrier factor nanosheet solution; in step 4, the cross-linking and composite process involves incubating at 4°C for 12-48 hours.

5. The preparation method according to claim 1, characterized in that, Titanium carbide nanosheet solutions were prepared by etching and exfoliation.

6. The preparation method according to claim 1, characterized in that, In step 5, the cross-linked composite microparticle suspension is mixed with the stem cell suspension and then seeded in a well plate, centrifuged, and cultured at 37°C for 12-72 hours. The well plate is a low-adhesion U-shaped 96-well plate.

7. A self-assembled stem cell material composite sphere, prepared by any one of claims 1 to 6.

8. The application of the self-assembled stem cell material composite spheres according to claim 7 in the preparation of materials for treating intervertebral disc degeneration.

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