Stem cell aggregate-derived decellular extracellular matrix and its application in bone defect repair

Abstract

The application relates to the technical field of biomaterials, in particular to a stem cell aggregate-derived decellular extracellular matrix and application thereof in bone defect repair. The decellular extracellular matrix is formed by subjecting a 3D cell aggregate in the form of a sphere to decellularization treatment, and the 3D cell aggregate is induced to form by transferring adult bone marrow mesenchymal stem cells after culture to a low-absorption cell culture carrier. The 3D dECM has a complete three-dimensional scaffold structure and abundant pores, while retaining key extracellular matrix components such as collagen, fibronectin and laminin, and is closer to the in-vivo tissue microenvironment; the 3D dECM significantly enhances the migration ability of cells, effectively improves the stemness of BMSCs, promotes extracellular matrix synthesis, and improves the mitochondrial function of BMSCs, has excellent cell regulation ability and functional activity, and thus solves the problems of low bioactivity and insufficient bionics of traditional 2D dECM.

Description

Technical Field

[0001] This invention relates to the field of biomaterials technology, specifically to a stem cell aggregate-derived acellular extracellular matrix (3D dECM) and its application in bone defect repair. Background Technology

[0002] With an aging population and an increasing number of osteoporosis patients, the incidence of bone defects is on the rise, and the repair of critical bone defects remains one of the most important challenges in the field of bone tissue engineering. Critical bone defects involve damage sizes far exceeding the body's own repair limits, making structural and functional reconstruction impossible through natural healing. This often leads to serious complications such as nonunion, delayed healing, and osteonecrosis, severely impacting patients' quality of life. Currently, clinical treatment of bone defects primarily relies on bone grafting and tissue engineering scaffolds, including autologous, allogeneic, xenogeneic bone grafts and synthetic bone substitutes. However, all these methods have numerous limitations and fail to meet the clinical demand for efficient, safe, and minimally invasive repair.

[0003] The extracellular matrix (ECM) is a complex network of biomolecules synthesized and secreted by cells, mainly consisting of collagen, fibronectin, laminin, and various bound growth factors. It provides physical support and a three-dimensional microenvironment for cells and regulates cell behavior through integrin signaling pathways, making it the most physiologically similar natural biological scaffold. Stem cell-derived ECM retains stem cell regeneration-related signals, promoting osteogenic differentiation and angiogenesis, inhibiting osteoclast activation, and regulating the inflammatory microenvironment, demonstrating significant advantages in bone tissue regeneration. For example, the injectable bone marrow mesenchymal stem cell extracellular matrix / agarose composite hydrogel disclosed in patent application number CN201410584721.1 can utilize the components within the ECM to provide nutritional support for the proliferation and differentiation of cells homing to the defect area. The extracellular matrix hydrogel microspheres disclosed in patent application number CN202410775843.2 can significantly promote in vitro osteogenic activity of bone marrow mesenchymal stem cells and inhibit osteoclast differentiation.

[0004] However, most existing ECMs are derived from traditional 2D monolayer adherent cell culture, which has many problems: First, 2D culture cannot simulate the three-dimensional environment in vivo, resulting in rapid loss of cell stemness and decreased matrix synthesis and secretion capacity. Secondly, 2D dECM has a two-dimensional structure, simple composition, low content, poor pore size and connectivity, and low biomimicry.

[0005] Third, existing decellularization processes easily damage the ECM structure, resulting in the loss of its active ingredients and a decrease in biocompatibility.

[0006] Compared to 2D monolayer adherent culture, 3D cell aggregates can activate glycolytic metabolic pathways and inhibit oxidative phosphorylation, forming a "high-energy state" more suitable for tissue regeneration. However, there is currently a lack of standardized, scalable 3D ECM preparation processes, and the molecular mechanism by which it promotes bone-angiogenic synergistic regeneration is unclear, limiting its clinical application. Furthermore, existing bone regeneration materials struggle to simultaneously achieve multiple functions such as "high biomimicry, high activity, pro-angiogenesis, immunomodulation, and minimally invasive injectability," failing to meet the repair needs of complex clinical scenarios such as critical bone defects. Summary of the Invention

[0007] The purpose of this invention is to provide a stem cell aggregate source derived from extracellular matrix, which uses a low-adsorption cell culture carrier to induce the formation of stem cell aggregates, and then prepares highly bioactive 3D dECM under standardized conditions to achieve vascular-bone synergistic regeneration and inflammatory microenvironment remodeling.

[0008] Another object of the present invention is to provide the application of the decellularized extracellular matrix derived from the above-mentioned stem cell aggregates in the repair of bone defects.

[0009] To achieve the above objectives, the present invention provides the following technical solution: a decellularized extracellular matrix derived from stem cell aggregates, wherein the decellularized extracellular matrix is ​​formed by decellularizing spherical 3D cell aggregates, and the 3D cell aggregates are induced to form by transferring adult bone marrow mesenchymal stem cells into a low-adsorption cell culture carrier after culture.

[0010] Furthermore, the average particle size of the decellularized extracellular matrix is ​​any value between 50 μm and 500 μm.

[0011] Furthermore, the decellularized extracellular matrix has a three-dimensional scaffold structure and retains collagen, fibronectin, and laminin.

[0012] This application also provides a method for preparing the above-mentioned decellularized extracellular matrix, comprising the following steps: S1. Adult bone marrow mesenchymal stem cells (BMSCs) were injected at a rate of 2000 / cm². 2 -4000 / cm 2 Cells were seeded at a density of [specific value] on cell culture dishes and cultured using complete cell culture medium; S2. Once the cell density reaches 90%, the cells are transferred to a low-adsorption cell culture carrier for further culture, inducing the formation of spherical 3D cell aggregates. S3. Collect the 3D cell aggregates and decellularize them, then incubate them with DNase I and wash them to obtain the decellularized extracellular matrix.

[0013] Furthermore, in step S1, growth factors or antibiotics are added to the complete cell culture medium to enhance cell activity.

[0014] Furthermore, in step S2, the low-adsorption cell culture carrier is a cell spheroid honeycomb culture sheet to improve the spheroid formation rate and uniformity; The cell density on each of the aforementioned cell sphere honeycomb culture sheets is 300,000-500,000 per sheet.

[0015] Further, in step S3, decellularization is performed using a buffer solution comprising Triton X-100 and ammonium hydroxide.

[0016] This application also provides the application of the above-mentioned decellularized extracellular matrix in bone defect repair.

[0017] Furthermore, the decellularized extracellular matrix is ​​directly injected into the bone defect site.

[0018] The beneficial effects of this invention are as follows: The 3D decellularized extracellular matrix derived from stem cell aggregates provided in this application is prepared by culturing bone marrow mesenchymal stem cells and then transferring them to a low-adsorption cell culture carrier to induce the formation of spherical 3D cell aggregates. Combined with standardized decellularization processing, a three-dimensional scaffold-like structure is created. This 3D dECM possesses a complete three-dimensional scaffold structure and abundant pores, while retaining key extracellular matrix components such as collagen, fibronectin, and laminin.

[0019] Compared to traditional 2D dECM, 3D dECM is significantly superior in terms of structural integrity and component diversity, and more closely resembles the in vivo tissue microenvironment. Furthermore, this 3D dECM significantly enhances cell migration ability, effectively improves the stemness of BMSCs, promotes extracellular matrix synthesis, and improves mitochondrial function of BMSCs, exhibiting excellent cell regulation capabilities and functional activity. This overcomes the problems of low bioactivity and insufficient biomimicry of traditional 2D dECM. Simultaneously, the preparation method of this 3D decellularized extracellular matrix is ​​highly reproducible and controllable, enabling large-scale production and overcoming the limitations of traditional two-dimensional ECM and non-standardized 3D dECM preparation.

[0020] This 3D dECM not only possesses osteogenic capabilities but also modulates angiogenesis-related factors, significantly enhancing HUVEC migration and vascular lumen formation, achieving bone-vascular synergistic regeneration, and providing a natural and more bioactive microenvironment for the repair of critical bone defects. Furthermore, this 3D dECM has a three-dimensional scaffold structure, allowing for direct application to bone defect repair via minimally invasive injection, without the need for loading onto microspheres or combining with other substances to form injectable hydrogels, demonstrating excellent clinical operability and translational potential.

[0021] The above description is merely an overview of the technical solution of the present invention. In order to better understand the technical means of the present invention and to implement it in accordance with the contents of the specification, the preferred embodiments of the present invention are described in detail below with reference to the accompanying drawings. Attached Figure Description

[0022] Figure 1 This is a schematic diagram of the preparation process of 3D dECM and 2D dECM shown in Embodiments 1 and 2 of the present invention; Figure 2 These are topographic images of the 3D dECM and 2D dECM shown in Embodiments 1 and 2 of the present invention; Figure 3 This is a fluorescence diagram of the 3D cell aggregates, 3D dECM, 2D cells, and the main components of 2D dECM shown in Examples 1 and 2 of the present invention. Figure 4 This is a comparison chart of the DNA content, VEGF content, collagen content and GAG content in the cell aggregates, 3D dECM, 2D cells and 2DdECM shown in Examples 1 and 2 of the present invention. Figure 5 This is a representative image of the cytokine antibody array membranes of 3D dECM and 2D dECM shown in Examples 1 and 2 of the present invention; Figure 6 The diagrams shown in Examples 1 and 2 of this invention illustrate the co-culture of 3D dECM and 2D dECM with BMSCs and the effects of different dECM materials on the viability of BMSCs cells. Figure 7 This is a comparative diagram showing the mitochondrial function of BMSCs after the 3D dECM and 2D dECM shown in Examples 1 and 2 were used to intervene in BMSCs under different conditions according to the present invention. Figure 8 The diagrams shown in Examples 1 and 2 of this invention illustrate the intervention of 3D dECM and 2D dECM in BMSCs for in vitro osteogenic differentiation, as well as the gross and optical microscope images stained with alizarin red. Figure 9 This is a comparison of the expression levels of osteogenic-related genes after 7 days and 21 days of intervention with 3D dECM and 2D dECM in osteogenic differentiation of BMSCs, as shown in Examples 1 and 2 of this invention. Figure 10 The images show a comparison of scratch closure in HUVECs treated by 3D dECM and 2D dECM as shown in Examples 1 and 2 of this invention. Figure 11The images show a comparison of the formation of tubular structures in cells during in vitro angiogenesis experiments using 3D dECM and 2D dECM, as shown in Examples 1 and 2 of this invention. Figure 12 Comparison of crystal violet staining results in in vitro angiogenesis experiments using 3D dECM and 2D dECM as shown in Examples 1 and 2 of this invention; Figure 13 This is a schematic diagram showing how 3D dECM and 2D dECM were respectively fabricated into 3D dECM hydrogels and 2D dECM hydrogels in Examples 1 and 2 of the present invention; Figure 14 The 3D dECM and 2D dECM shown in Examples 1 and 2 of this invention were respectively filled into the round defects of the mouse skull. The gross images of the skull samples taken 4 weeks after the operation and the results of Micro-CT scans are shown. Figure label: 1. Adherent cells; 2. Semi-permeable membrane; 3. Culture medium containing dECM. Detailed Implementation

[0023] The technical solutions of the present invention will now be clearly and completely described with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention. Furthermore, the technical features involved in the different embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other.

[0024] A preferred embodiment of this application illustrates a stem cell aggregate-derived decellularized extracellular matrix, which is formed from spherical 3D cell aggregates after decellularization. These 3D cell aggregates are induced by transferring cultured adult bone marrow mesenchymal stem cells into a low-adsorption cell culture carrier. In this embodiment or other embodiments, the average particle size of the decellularized extracellular matrix is ​​distributed in the range of 50 μm to 500 μm, exhibiting micron-scale structural characteristics.

[0025] In one embodiment, the decellularized extracellular matrix has a three-dimensional scaffold structure, meaning that it retains the natural three-dimensional scaffold structure and maintains a highly cross-linked network of fibers in space. Simultaneously, the decellularized extracellular matrix retains key extracellular matrix proteins such as collagen, fibronectin, and laminin, making it suitable for biomedical applications such as bone regeneration, angiogenesis, and tissue engineering.

[0026] The decellularized extracellular matrix provided in one embodiment is prepared by the following steps: S1, Adult bone marrow mesenchymal stem cells were administered at a rate of 2000 / cm². 2 -4000 / cm 2 Cells were seeded at a density of [specific value] on cell culture dishes and cultured using complete cell culture medium; S2. Once the cell density reaches 90%, the cells are transferred to a low-adsorption cell culture carrier for further culture, inducing the formation of spherical 3D cell aggregates. S3. Collect 3D cell aggregates and decellularize them. Then, incubate them with DNase I and wash them to obtain decellularized extracellular matrix, i.e., 3D dECM.

[0027] In step S1, BMSCs are cultured under suitable density and nutrient conditions to ensure rapid proliferation and obtain a sufficient number of cells for subsequent 3D cell aggregate formation. The complete cell culture medium used includes 89% α-MEM, 10% fetal bovine serum, and 1% penicillin-streptomycin. This complete culture medium provides essential nutrients, serum support, and antibiotic protection, ensuring cell health and intact biological activity. Appropriate cell density avoids either slow proliferation due to excessive sparseness or early differentiation due to excessive density, laying the foundation for subsequent spheroid formation.

[0028] In step S2, the low-adsorption cell culture carrier reduces cell adhesion to the culture vessel, promoting spontaneous aggregation of BMSCs to form spherical 3D cell aggregates. These 3D cell aggregates produce a more biomimetic 3D extracellular matrix, i.e., a three-dimensional extracellular matrix with excellent three-dimensionality. Compared to the 2D extracellular matrix generated by 2D plate culture, this three-dimensional extracellular matrix more closely resembles the in vivo tissue microenvironment.

[0029] In step S3, decellularization of the spherical 3D cell aggregates preserves their structural features and major proteins, maintaining their biological activity while removing cellular components and effectively reducing immunogenicity. The resulting storable extracellular matrix (ECM) after decellularization is crucial for the direct use of stem cell aggregates (ECM) as an injectable material. Co-incubation of the decellularized ECM with DNase I effectively removes residual nucleic acids, further reducing immunogenicity and ensuring its safety for in vivo application. Repeated washing with buffer removes residual chemical reagents, protecting the ECM protein structure and function. The prepared 3D dECM can be stored short-term at 4°C; if not used immediately, it should be transferred to -80°C for long-term storage.

[0030] In one embodiment, in step S1, growth factors or antibiotics are added to the complete cell culture medium to enhance cell activity.

[0031] In one embodiment, in step S2, the low-adsorption cell culture carrier is typically a commercially available cell spheroid honeycomb culture plate, such as the cell spheroid honeycomb culture plate purchased from Suzhou Tri-Bio Technology Co., Ltd., catalog number AUT042801, to improve the spheroid formation rate and uniformity. In this embodiment or other embodiments, the cell density on each cell spheroid honeycomb culture plate is preferably any value between 300,000 / plate and 500,000 / plate, such as 400,000 cells / plate. By controlling the number of cells per plate, the spheroid size and structure can be made more uniform, facilitating subsequent processing and 3D dECM quality control.

[0032] In one embodiment, in step S3, a buffer solution comprising Triton X-100 and ammonium hydroxide is used for decellularization.

[0033] This application also provides the application of the aforementioned decellularized extracellular matrix in bone defect repair. In this embodiment or other embodiments, the decellularized extracellular matrix can be directly injected into the bone defect site. This decellularized extracellular matrix can promote new bone formation, angiogenesis and tissue regeneration, improve biomechanical properties, and exhibit good in vivo biocompatibility, providing an ideal scaffold material for bone-vascular synergistic regeneration.

[0034] Example 1

[0035] S1. Obtain adult bone marrow mesenchymal stem cells and administer them at a rate of 3000 / cm³. 2 Cells were seeded at a density of 89% on cell culture dishes and cultured in a complete cell culture medium containing 89% α-MEM, 10% fetal bovine serum, and 1% penicillin-streptomycin at 37°C and 5% CO2.

[0036] S2. After the cell density reaches 90%, Figure 1 As shown in (b), the adult bone marrow mesenchymal stem cells were transferred to a low-adsorption cell spheroid cell culture plate at a cell density of approximately 400,000 cells / plate for culture, which promoted the spontaneous formation of cells into spheroids and induced the formation of spherical 3D cell aggregates.

[0037] S3. Collect the above-mentioned 3D cell aggregates and place them in a buffer solution (PBS) containing 0.5% (v / v) Triton X-100 and 20 mM ammonium hydroxide. Incubate at 37°C for 10 minutes for decellularization. Subsequently, wash the incubated extracellular matrix three times with buffer to remove cellular components while retaining the fibrous structure and protein components in the ECM, obtaining a safe, structurally intact, and functionally active extracellular matrix. Then, at 37°C, co-incubate the decellularized extracellular matrix with 1 KU / mL DNase I for 1 hour, and wash twice with buffer to obtain a three-dimensional scaffold-like decellularized extracellular matrix, i.e., 3D dECM. Store the decellularized extracellular matrix at 4°C for later use.

[0038] Example 2

[0039] like Figure 1 As shown in (a), adult bone marrow mesenchymal stem cells (BMSCs) were obtained and quantified at a concentration of 3000 / cm³. 2 Cells were seeded at a density suitable for their formation onto cell culture dishes and cultured in a complete cell culture medium containing 89% α-MEM, 10% fetal bovine serum, and 1% penicillin-streptomycin at 37°C and 5% CO2. After 5 days of culture, 0.5% (v / v) Triton X-100 and 20 mM ammonium hydroxide in a buffer solution were added to the cell culture dishes, and the cells were incubated at 37°C for 5 minutes for decellularization. The 2D cells were then washed three times with buffer to remove cellular components. Next, the decellularized extracellular matrix was co-incubated with 100 U / mL DNase I at 37°C for 10 minutes, followed by two washes with buffer to obtain a fibrous network structure of 2D dECM, i.e., 2D decellularized extracellular matrix. This 2D dECM was then stored at 4°C for later use.

[0040] The morphology and structure of the 3D dECM prepared in Example 1 and the 2D dECM prepared in Example 2 were observed under a 40x objective lens using an excitation confocal microscope. Simultaneously, to further analyze their microstructure, the surface microstructure of the 2D dECM and 3D dECM was examined using a scanning electron microscope (SEM). Specifically, the 2D dECM and 3D dECM were fixed with 2.5% glutaraldehyde at room temperature for 10 min; then, they were subjected to gradient ethanol dehydration (gradients of 50%, 75%, 80%, 95%, and 100%) and supercritical point drying; subsequently, the 2D dECM and 3D dECM samples were fixed on a stage using conductive adhesive, and gold sputtering was performed on the samples to obtain SEM images. The results are as follows: Figure 2 As shown.

[0041] Depend on Figure 2 As shown in (a) and (b), the 2D dECM prepared in Example 2 has a uniform fiber network distribution, with its fibers mainly spreading along the plane, resulting in limited spatial support and an inability to provide complete three-dimensional support. However, from... Figure 2 As shown in (c) and (d), the 3D dECM prepared in Example 1 possesses abundant porosity and a crisscrossing three-dimensional network fiber structure, enabling it to form a three-dimensional scaffold. Therefore, the 3D dECM provided in Example 1 exhibits a complete three-dimensional spatial structure, more closely resembling the in vivo tissue microenvironment. This contributes to enhancing cell adhesion, migration, and osteogenic potential, resulting in significant advantages in tissue engineering and bone regeneration applications.

[0042] To evaluate the protein retention and spatial structure of 2D cells, 2D dECMs, 3D cell aggregates, and 3D dECMs, immunofluorescence staining, proteomics analysis, functional factor analysis, and immunogenicity were used for characterization and analysis. Specific procedures were as follows: 2D cells and 2D dECMs, as well as 3D cell aggregates and 3D dECMs, were fixed with 4% paraformaldehyde for 20 min. After fixation, the fixative was removed, and the cells were washed three times with buffer. Subsequently, the cells were blocked with 1% bovine serum albumin (BSA) for 1 h at room temperature, followed by incubation with primary antibodies against fibronectin, collagen, and laminin (dilution ratio 1:500) at 4°C for 12 h. Then, the incubated 2D cells and 2D dECMs, as well as 3D cell aggregates and 3D dECMs, were washed twice with buffer. After two washes, Alexa Fluor 488 or 594 fluorescent secondary antibodies (dilution ratio 1:500) were added, and the cells were incubated at room temperature for 1 h. Finally, the cells were washed three times with buffer, counterstained with a benzimidazole fluorescent dye (Hoechst), and then imaged using a confocal microscope. 2D cells and 2D dECM, 3D cell aggregates and 3D dECM samples were lysed using strong RIPA lysis buffer, and the total protein concentration was determined using the BCA protein assay. Next, the protein concentrations of 2D cells and 2D dECM, 3D cell aggregates and 3D dECM samples were standardized, requiring a total protein loading of at least 50 μg. Then, the cytokine content in 3D dECM and 2D dECM was detected using a human cytokine antibody array membrane (120 target sites, ab193656, abcam). The signal intensity of each cytokine was quantified using ImageJ software and standardized using the positive control on each membrane. Results are as follows: Figure 3 , Figure 4 and Figure 5 As shown.

[0043] Depend on Figure 3 It was found that the DNA content in 2D dECM and 3D dECM was significantly lower than that in their corresponding 2D cells and 3D cell aggregates. Specifically, 2D cells contained DNA signaling, and their fibronectin and collagen were evenly distributed; while in 2D dECM obtained after decellularization, the DNA signaling was significantly reduced, although fibronectin and collagen remained evenly distributed. Compared to 3D cell aggregates, most of the DNA in the decellularized 3D dECM was removed, but abundant ECM proteins, such as fibronectin, collagen, and laminin, were retained. Furthermore, 3D dECM retained its three-dimensional scaffold structure. Therefore, 3D dECM obtained after decellularization retains the main ECM proteins while exhibiting excellent decellularization effect, significantly reduced immunogenicity, and intact three-dimensional structure, thus preserving its biological activity. Figure 4 It is evident that, compared to 2D dECM, 3D dECM has a richer proteome, containing more extracellular matrix proteins, including collagen I and III, fibronectin, and laminin. This indicates that 3D dECM is rich in proteins related to osteoogenesis, angiogenesis, and immune regulation, suggesting higher potential for biological activity. Figure 5 It was found that, compared to 2D dECM, 3D dECM retained a higher overall level of various key functional growth factors and cytokines, such as PDGF-BB, TGF-B3, IGF-1, GDNF, and BDNF. This indicates that it has a better ability to retain bioactive molecules during decellularization. Meanwhile, while some functional factors in 2D dECM showed varying degrees of decline, 3D dECM maintained a relatively rich and diverse spectrum of signaling molecules. This suggests that 3D dECM, in its three-dimensional structural state, has a better "protective effect" and spatial retention advantage on the extracellular matrix microenvironment and soluble factors. Comprehensive morphological, proteomic, and functional factor analyses show that 3D dECM possesses excellent three-dimensional structural integrity and, compared to 2D dECM, exhibits higher levels of protein richness and functional factor retention, demonstrating greater biological activity and clinical application potential, especially suitable for bone-vascular synergistic regeneration.

[0044] To evaluate the functional regulatory effects of the 3D dECM prepared in Example 1 and the 2D dECM prepared in Example 2 on bone marrow mesenchymal stem cells (BMSCs), 3D dECM and 2D dECM were co-cultured with BMSCs, and the expression changes of genes related to cell stemness and mitochondrial function were detected by RT-qPCR. Specific procedures are as follows: Figure 6As shown in (a), BMSCs were seeded into a 24-well cell culture chamber (brand: LABSELECT, catalog number: 14342) and cultured to allow the BMSCs to adhere and form adherent cells 1. Subsequently, dECM-containing medium 3 (containing either 3D dECM or 2D dECM) was added. The semi-permeable membrane 2 in the dECM-containing medium 3 contacted the adherent cells 1, and co-cultured. A control group consisting of BMSCs without any dECM was also included. OD values ​​were measured on day 1 (D1), day 3 (D3), day 5 (D5), and day 7 (D7) of co-culture. 450 The values ​​were used to assess the impact of different dECM treatments on the functional activity of BMSCs. The detection results are as follows: Figure 6 As shown in (b). On day 3 of co-culture, cell samples were collected, total RNA was extracted, and RT-qPCR analysis was performed on stemness-related genes (such as PGC-1α) and mitochondrial function-related genes (such as ATP5A, COX4, ND4, CAT, etc.). The results are shown in Figure 1. Figure 7 As shown.

[0045] Depend on Figure 6 As shown in (b), the OD450 values ​​of the three groups of BMSCs did not differ significantly during co-culture, indicating that dECM treatment did not negatively affect the functional activity of BMSCs. Figure 7 As shown in (a), compared with the control group and 2D dECM, 3D dECM significantly enhanced the expression of stemness-related genes and mitochondrial function genes in BMSCs, demonstrating superior stemness maintenance and mitochondrial function enhancement capabilities. Furthermore, from Figure 7 As shown in Figure (b), under simulated inflammation conditions, 3D dECM significantly protects the mitochondrial function of BMSCs, effectively reducing the inhibitory effects of inflammation on cellular metabolism and energy production. This indicates that 3D dECM not only retains abundant ECM proteins and functional factors, but also enhances the cellular function and mitochondrial activity of BMSCs by providing a more in vivo three-dimensional microenvironment, achieving an anti-inflammatory protective effect. Its bioactivity potential is superior to 2D dECM, and it has significant application value in bone tissue regeneration and functional repair.

[0046] To evaluate the regulatory effects of the 3D dECM prepared in Example 1 and the 2D dECM prepared in Example 2 on osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs), an in vitro osteogenic differentiation experiment was designed. Specific procedures are as follows: Figure 8As shown in (a), BMSCs were seeded into a 24-well plate cell culture system to form adherent cells 1. Medium 3 containing dECM (3D dECM or 2D dECM) was added to the 24-well plate cell culture chambers. The dECM in the medium 3 permeated through a semi-permeable membrane 2 to contact and intervene in the adherent cells 1. Subsequently, after the cell density reached 80%, osteogenic induction solution was added to the culture medium, and the cells were cultured for 7 and 21 days under the condition of adding osteogenic induction solution. Samples were collected for analysis. Alizarin Red S staining was used to detect the formation of mineralized nodules in the cells. Simultaneously, cellular RNA was extracted, and the expression levels of osteogenic differentiation-related genes, including early marker genes RUNX2, COL1A1, and ALP, and late marker genes OCN, OPN, and RUNX2, were detected by RT-qPCR. The detection results are shown below. Figure 8 , Figure 9 As shown.

[0047] Depend on Figure 8 As shown in (b), BMSCs treated with 3D dECM exhibited a significantly increased and more uniformly distributed mineralized nodules, demonstrating a higher degree of mineralization compared to BMSCs treated with 2D dECM. This indicates that 3D dECM can significantly promote osteogenic differentiation of cells. Figure 9 As shown in Figure (a), compared with BMSCs after 2D dECM intervention and the control group, BMSCs after 3D dECM intervention showed significantly higher expression levels of factors such as RUNX2, COL1A1, and ALP. This indicates that 7 days of culture can significantly enhance the expression of osteogenic-related genes. Meanwhile, Figure 9 As shown in Figure (b), the expression of osteogenic-related genes was significantly enhanced after 21 days of culture. These osteogenic-related genes include RUNX2, OCN, and OPN. This indicates that the expression of osteogenic differentiation-related genes RUNX2, COL1A1, ALP, OCN, and OPN was significantly upregulated in BMSCs after 3D dECM intervention, showing a simultaneous enhancement of early and late osteogenic markers. This further demonstrates that 3D dECM, through the combined action of a three-dimensional structural scaffold and preserving functional factors, significantly enhances the osteogenic potential of BMSCs, providing an excellent in vitro platform for promoting osteogenic differentiation in bone tissue engineering.

[0048] To evaluate the regulatory effects of the 3D dECM prepared in Example 1 and the 2D dECM prepared in Example 2 on the migration and angiogenesis of vascular endothelial cells (HUVECs), an in vitro vascular function experiment was designed. The scratch assay was performed as follows: HUVECs were seeded in 6-well plates and cultured until confluence. A straight scratch was made in each well using the tip of a 200 μL pipette. After washing with PBS to remove detached cells, the cells were placed in a scaffold leaching solution containing either 3D dECM or 2D dECM for culture. Cell migration was observed after 24 hours of culture. The closure of the scratch was photographed using a microscope, and quantitative analysis was performed using ImageJ software. The results are as follows: Figure 10 As shown. The specific procedure for the in vitro angiogenesis assay (Tube Formation Assay) is as follows: HUVECs are seeded on Matrigel pre-coated plates, and culture medium containing 3D dECM or 2D dECM is added. After culturing for 6-12 hours, the formation of tubular structures in the cells is observed, and the results are shown in the figure. Figure 11 As shown in the diagram, HUVECs were spread onto the upper layer of semipermeable membrane 2 in a 24-well cell culture chamber. Culture medium containing 3D dECM or 2D dECM was placed below the semipermeable membrane. Attracted by the 3D or 2D dECM, the HUVECs migrated through the semipermeable membrane and adhered. The migration ability of ECM to cells was then assessed using a crystal violet staining assay, and the results are shown below. Figure 12 As shown.

[0049] Depend on Figure 10 It can be seen that HUVECs treated with 3D dECM migrated significantly faster than those treated with 2D dECM and the control group, with a significantly improved scratch closure rate, demonstrating a stronger ability to promote migration. Figure 11 It can be seen that HUVECs treated with 3D dECM form a more complete tubular network with more nodes and a significantly increased tube length, demonstrating a stronger pro-angiogenic capacity compared to HUVECs treated with 2D dECM. Figure 12 As shown, crystal violet staining further confirmed the promoting effect of 3D dECM on HUVEC migration and angiogenesis. Therefore, 3D dECM, through its three-dimensional scaffold structure and retained functional factors, can mimic the in vivo microenvironment, enhance vascular endothelial cell function, and provide an ideal scaffold material for bone-vascular synergistic regeneration.

[0050] To verify the function of the 3D dECM prepared in Example 1 in promoting bone repair in vivo, a mouse skull defect model was constructed, and imaging and histological analyses were performed. The specific procedures for constructing the mouse skull defect model were as follows: Preoperative preparation for skull defect surgery: Isoflurane inhalation anesthesia was performed (induction concentration 2%, maintenance concentration 0.4%), the hair on the top of the head was shaved, and the area was disinfected three times alternately with povidone-iodine and 75% ethanol.

[0051] Bone defect surgery: An incision of approximately 1 cm is made along the midline of the skull (sagittal suture). The skin and soft tissues are dissected layer by layer until the skull is clearly exposed. Under continuous irrigation with saline solution, a 3 mm diameter circular defect is drilled in the center of the skull using a miniature ring-shaped diamond drill bit (2 mm inner diameter, 3 mm outer diameter, rotation speed ≤1000 rpm), penetrating only the outer table and diploic plate, preserving the inner table and dura mater to avoid brain injury. Postoperatively, a sponge is used for gentle pressure to stop bleeding and prevent brain tissue damage.

[0052] Material filling and postoperative treatment: The 3D dECM prepared in Example 1 and the 2D dECM prepared in Example 2 were placed in centrifuge tubes and centrifuged at 3500 rpm. After centrifugation for 8 minutes, they were placed at -80℃ overnight and then thawed. Figure 13 As shown, 3D dECM hydrogel and 2D dECM hydrogel were obtained respectively. The 3D dECM hydrogel and 2D dECM hydrogel were directly injected into the circular defect, and commercially available GelMa material was then used to fill the defect. A treatment group (the defect group) was also established without any repair material. The incision was then sutured layer by layer and disinfected again. Antibiotics were added to the patient's drinking water for three days post-surgery to prevent infection.

[0053] At week 4 post-surgery, all mice were euthanized and their skulls were completely removed. Micro-CT scans were used to analyze the skull samples (65 kV, 385 μA, 18 μm resolution), and the bone value (BV), total value (TV), newly formed bone trabecular volume fraction (BV / TV), and bone trabecular thickness (Tb.Th, in mm) were calculated. The results are as follows: Figure 14 As shown.

[0054] Depend on Figure 14 It was found that at 4 weeks post-surgery, the volume of newly formed trabecular bone in the skull defect area of ​​the 3D dECM group was significantly larger than that of the 2D dECM group and the GelMa material group, and the BV / TV ratio was significantly increased. Simultaneously, the thickness of newly formed trabecular bone in the skull defect area of ​​the 3D dECM group was significantly greater than that of the 2D dECM group and the GelMa material group. This indicates that 3D dECM can significantly promote new bone formation in skull defects in mice. Furthermore, this demonstrates that the 3D dECM provided in this application has excellent bone repair potential in vivo and can serve as a superior material for bone tissue engineering and bone regeneration.

[0055] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0056] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims.

Claims

1. A stem cell aggregate derived from decellularized extracellular matrix, characterized in that, The decellularized extracellular matrix is ​​formed by decellularizing spherical 3D cell aggregates, which are induced to form by transferring adult bone marrow mesenchymal stem cells into a low-adsorption cell culture carrier after culture.

2. The decellularized extracellular matrix as described in claim 1, characterized in that, The average particle size of the decellularized extracellular matrix is ​​any value between 50 μm and 500 μm.

3. The decellularized extracellular matrix as described in claim 1, characterized in that, The decellularized extracellular matrix has a three-dimensional scaffold structure and retains collagen, fibronectin, and laminin.

4. The method for preparing decellularized extracellular matrix according to any one of claims 1-3, characterized in that, Includes the following steps: S1, Adult bone marrow mesenchymal stem cells were administered at a rate of 2000 / cm². 2 -4000 / cm 2 Cells were seeded at a density of [specific value] on cell culture dishes and cultured using complete cell culture medium; S2. Once the cell density reaches 90%, the cells are transferred to a low-adsorption cell culture carrier for further culture, inducing the formation of spherical 3D cell aggregates. S3. Collect the 3D cell aggregates and decellularize them, then incubate them with DNase I and wash them to obtain the decellularized extracellular matrix.

5. The preparation method according to claim 4, characterized in that, In step S1, growth factors or antibiotics are added to the complete cell culture medium to enhance cell activity.

6. The preparation method according to claim 4, characterized in that, In step S2, the low-adsorption cell culture carrier is a cell spheroid honeycomb culture sheet to improve the spheroid formation rate and uniformity; The cell density on each of the aforementioned cell sphere honeycomb culture sheets is 300,000-500,000 per sheet.

7. The preparation method according to claim 4, characterized in that, In step S3, decellularization is performed using a buffer solution containing Triton X-100 and ammonium hydroxide.

8. The application of the decellularized extracellular matrix according to any one of claims 1-3 in bone defect repair.

9. The application as described in claim 8, characterized in that, The decellularized extracellular matrix is ​​injected directly into the bone defect site.

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