Application of apoptotic cell pretreated macrophages in preparation of osteoarthritis treatment preparation, cell preparation and preparation method

Abstract

The invention provides application of apoptotic cell pretreated macrophages in preparation of an osteoarthritis treatment preparation, a cell preparation and a preparation method, and relates to the technical field of biomedicine.In the embodiment of the invention, based on a mouse development model and an osteoarthritis (OA) model, it is found that in the area away from the cartilage surface, the apoptotic cell pretreated macrophages are subjected to apoptotic cell pretreatment, and the apoptotic cell pretreated macrophages are subjected to apoptotic cell pretreatment in the area away from the cartilage surface; in the macrophages, the content of the interment-related LAMP1 protein is obviously higher than that of the macrophages close to the cartilage surface area, and the expression results of TGF-beta 1 and LRG1 are respectively opposite and consistent with those of the LAMP1. The invention also finds that after the chemotactic effect of apoptotic cells on macrophages is weakened, the cellular burial effect is limited, and the cartilage matrix development disorder is caused. Based on development process inspiration, the invention provides a treatment strategy: bone marrow-derived macrophages pretreated by apoptotic cells are injected into articular cavities, and a good cartilage regeneration treatment effect can be achieved in a mouse arthritis model. In addition, the bone marrow-derived macrophages pretreated by apoptotic cells can still express TGF-beta1 after being injected into the body for 3 days, so that the effect of continuously promoting cartilage regeneration is achieved.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to the use of macrophages pretreated with apoptotic cells in the preparation of a preparation for treating osteoarthritis, a cell preparation and a preparation method. Background Art

[0002] Synovial joints are highly flexible joints primarily found in the limbs. Their multidirectional motion and biomechanical properties enable them to perform complex movements while withstanding dynamic loads equivalent to 10 times their body weight. Synovial joints are composed of multiple components that work together to achieve their function, including the synovium, joint capsule, synovial fluid, and articular cartilage. Articular cartilage is a highly specialized tissue that lines the ends of the epiphyses within the synovial joint cavity. It primarily consists of type II collagen and glycosaminoglycans (GAGs) and is characterized by low cellularity. Due to its translucent appearance, articular cartilage is also known as hyaline cartilage. Articular cartilage lacks blood vessels, lymphatic vessels, or nerves. Its cells produce an extracellular matrix to maintain their environment, with minimal cell turnover. Glycosaminoglycans (including chondroitin sulfate and hyaluronic acid) bind to longitudinally oriented type II collagen via core proteins, forming a complex extracellular matrix network that absorbs and conducts mechanical forces to the articular cartilage. One of the key features of articular cartilage is its smooth surface, composed of lubricating proteins and horizontally oriented collagen, which reduces friction during bone movement.

[0003] Injury to articular cartilage often leads to joint dysfunction, such as cartilage defects and osteoarthritis (OA). Due to its avascular nature, articular cartilage lacks access to abundant nutrients or circulating progenitor cells, and its nearly acellular nature, lacks the innate ability to mount an adequate healing response. To achieve functional regeneration of damaged or missing cartilage, artificial intervention to promote cartilage regeneration is often necessary. Current approaches to articular cartilage regeneration include medication, cell therapy—autologous chondrocyte implantation (ACI) and matrix-induced autologous chondrocyte implantation (MACI), autologous articular cartilage transplantation, and bone marrow stimulation. Cell therapy utilizes cells to reduce inflammation, optimize the microenvironment, and promote chondrogenic differentiation. While these approaches can alleviate symptoms and improve joint function, many unresolved challenges remain. Regenerated cartilage often degenerates and develops into fibrocartilage tissue, ultimately disappearing, leading to delamination and exposure of the subchondral bone. It is also difficult for regenerated cartilage to fully integrate with existing cartilage and subchondral bone, resulting in abnormal stress at the interface causing cell death and a reduction in surrounding normal chondrocytes. For large and deep defects, such as those in patients with severe osteoarthritis, existing technologies cannot cure them. In addition, the success rate of cartilage regeneration gradually decreases with the age of the patient. In short, in current research and clinical practice, the fundamental problem faced is how to regenerate permanent hyaline cartilage and how to "develop" articular bone that is consistent with the structure, composition, function and sustainability of the original articular cartilage. Summary of the Invention

[0004] The present invention provides the use of macrophages pretreated with apoptotic cells in the preparation of a preparation for treating osteoarthritis, a cell preparation and a preparation method, in order to at least partially solve the above problems.

[0005] A first aspect of the present invention provides a use of macrophages pretreated with apoptotic cells in the preparation of a preparation for the treatment of osteoarthritis. The macrophages pretreated with apoptotic cells are used to alleviate osteoarthritis inflammation and promote osteoarthritis cartilage regeneration, including promoting the expression of SOX9 and type II collagen in chondrocytes; in areas away from the cartilage surface, the content of LAMP1 protein associated with burial in macrophages is higher than that in macrophages in areas close to the cartilage surface, TGF-β1 expression is lower than that in macrophages in areas close to the cartilage surface, and LRG1 expression is higher than that in macrophages in areas close to the cartilage surface.

[0006] Optionally, the apoptotic cell-pretreated macrophages are obtained by co-culturing macrophages and apoptotic cells at a ratio of 1:1-10.

[0007] Preferably, the macrophages are bone marrow-derived macrophages, and the ratio of the bone marrow-derived macrophages to apoptotic cells is 1:5.

[0008] Optionally, the macrophage efferocytosis achieves a dynamic balance of cartilage differentiation through the LRG1 / TGF-β1 bidirectional signaling axis: bone marrow-derived macrophages are promoted to secrete TGF-β1 through efferocytosis, driving the increased expression of Sox9, Col2, and Acan and corresponding cartilage differentiation, while inhibiting the expression of the fibrosis marker Col1.

[0009] Alternatively, during joint development, macrophages in the central area away from the cartilage surface show high levels of burial, driving high expression of LRG1, maintaining the morphology of the joint cavity by inhibiting cartilage differentiation; in the marginal area close to the cartilage surface, macrophages secrete TGF-β1 when burializing apoptotic cells, promoting chondrogenic differentiation and matrix production of chondrogenic progenitor cells.

[0010] A second aspect of the present invention provides a macrophage preparation pretreated with apoptotic cells, which is used to prepare a preparation for treating osteoarthritis. The macrophages pretreated with apoptotic cells are used to alleviate osteoarthritis inflammation and promote osteoarthritis cartilage regeneration, including promoting the expression of SOX9 and type II collagen in chondrocytes; in areas away from the cartilage surface, the content of LAMP1 protein associated with burial in macrophages is higher than that in macrophages in areas close to the cartilage surface, TGF-β1 expression is lower than that in macrophages in areas close to the cartilage surface, and LRG1 expression is higher than that in macrophages in areas close to the cartilage surface.

[0011] Optionally, the apoptotic cell-pretreated macrophages are obtained by co-culturing macrophages and apoptotic cells at a ratio of 1:1-10.

[0012] The third aspect of the present invention provides a method for preparing the macrophage preparation pretreated with apoptotic cells as described in the second aspect, the method comprising: Macrophages and apoptotic cells are co-cultured according to a preset ratio to obtain a macrophage preparation pretreated with apoptotic cells.

[0013] Optionally, the method further includes: Add fetal bovine serum (FBS) and penicillin-streptomycin solution (PS) to α-MEM medium to a volume concentration of 10% FBS and 1% PS, respectively. Add macrophage colony-stimulating factor (M-CSF) to a final concentration of 50 ng / mL to obtain macrophage differentiation medium. Extract primary bone marrow cells and culture for about 8-12 hours. After the mesenchymal stem cells adhere to the wall, remove the unattached cells and transfer them to a regular culture dish. Add 10 mL of macrophage differentiation medium and culture in a cell culture incubator at 37°C and 5% CO2. After 7 days of continuous culture, the mononuclear precursor cells differentiated into macrophages, and bone marrow-derived macrophages were obtained; Transfer the Jurkat cell suspension to a centrifuge tube, centrifuge at 1200 rpm for 5 minutes, discard the supernatant, resuspend with PBS, and inoculate in a common culture dish to a cell inoculation density of 1×10 5 cells / mL; irradiated with 254 nm ultraviolet light for 15 minutes; and then incubated in PBS for 2-3 hours to obtain apoptotic cells.

[0014] Optionally, macrophages are co-cultured with apoptotic cells according to a preset ratio to obtain a macrophage preparation pretreated with apoptotic cells, comprising: Apoptotic cells were added to the BMDM culture dish in differentiation culture at a preset ratio. After incubation at 37°C for 6 hours, the supernatant was discarded. After washing once with PBS, the cells were digested with 0.25% trypsin at 37°C for 2 minutes. The culture dish was gently tapped to observe that all the cells were floating and not attached to the wall. After that, 4 mL of proliferation medium was added to terminate the digestion and the liquid was collected in a centrifuge tube. The collected cell suspension was centrifuged at 1000 rpm for 5 minutes and the supernatant was discarded. PBS was added to obtain a macrophage preparation pretreated with apoptotic cells.

[0015] In the examples of the present invention, based on mouse developmental models and osteoarthritis (OA) models, it was found that the level of LAMP1, a protein associated with efferocytosis, was significantly higher in macrophages distal to the cartilage surface than in macrophages proximal to the cartilage surface. The expressions of TGF-β1 and LRG1 were, respectively, opposite and consistent with the LAMP1 results. The present invention also found that the reduced chemotaxis of macrophages by apoptotic cells, leading to restricted efferocytosis, impaired cartilage matrix development. Inspired by this developmental process, the present invention proposes a therapeutic strategy: intra-articular injection of bone marrow-derived macrophages pretreated with an appropriate ratio of apoptotic cells can achieve significant cartilage regeneration in a mouse arthritis model. Furthermore, bone marrow-derived macrophages pretreated with an appropriate ratio of apoptotic cells continued to express TGF-β1 three days after injection, demonstrating a sustained promotion of cartilage regeneration. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] In order to more clearly illustrate the technical solution of the present invention, the following briefly introduces the drawings required for use in the description of the present invention. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0017] Figure 1 This is a schematic diagram of chondrogenesis-related gene expression in cells in the co-culture model provided in Example 1 of the present invention; Figure 2 Schematic diagram of the expression of cartilage-related genes in the co-culture model of Example 1 provided by the present invention, in which BMDM lacks CX3CR1; Figure 3 Schematic diagram of the effects of different ratios of BMDM and ACs on the expression of Lrg1 and Tgf-β1 in Example 1 provided by the present invention; Figure 4 Schematic diagram of the effect of different BMDM and ACs ratios on the expression of chondrogenesis-related genes in the co-culture model provided in Example 1 of the present invention; Figure 5 This is a schematic diagram of promoting osteoarthritis (OA) cartilage regeneration in BMDM pretreated with ACs in Example 2 provided by the present invention. DETAILED DESCRIPTION

[0018] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, the present invention is further described in detail below with reference to the accompanying drawings and specific embodiments.

[0019] Current models of articular cartilage development primarily focus on the development of cells along the cartilage lineage, including the fate and molecular mechanisms of stem cells, cartilage progenitor cells, and chondrocytes. This provides guidance and reference for existing major articular cartilage regeneration strategies. However, these strategies, based on existing developmental models, still face regenerative challenges. Consequently, more intraarticular cells involved in regulatory functions are being considered within regenerative strategies, such as macrophages, which reside in the synovium. As key immune cells, macrophages play an essential role in regulating the immune microenvironment during cartilage regeneration, particularly in osteoarthritis models based on abnormal inflammation. It is generally believed that the long-term presence of proinflammatory M1-like macrophages is detrimental to tissue repair, while anti-inflammatory M2-like macrophages can promote tissue regeneration. Injecting polarized M2-like macrophages in vitro or designing biomaterials that can modulate the macrophage phenotype toward a reparative phenotype have been shown to effectively achieve cartilage regeneration in osteoarthritis models.

[0020] The inventors' team's preliminary research found that in a cartilage defect model, depletion of macrophages in the joint hindered the regeneration of articular cartilage, resulting in an increase in apoptotic cells and a decrease in proliferating cells in the cartilage. Injection of macrophages with M1, M2a, or M2c phenotypes all contributed to cartilage regeneration, with M2c being the most effective. Although macrophages have been shown to play an important regulatory role in the process of cartilage regeneration and to have complex spatiotemporal regulation, it is difficult to correspond to existing cartilage development models. This is because the role of macrophages in existing cartilage development models remains to be clarified.

[0021] In summary, the inventors hypothesize that in developing synovial joints, in addition to clearing apoptotic cells during development (i.e., efferocytosis), macrophages also regulate cartilage precursor cell differentiation and matrix secretion through efferocytosis, promoting the production of prochondrogenic signals such as transforming growth factor-β1 (TGF-β1) and antichondrogenic signals such as leucine-rich α2-glycoprotein 1 (LRG1). This regulatory effect is spatiotemporal, with secretion levels varying at specific developmental times and anatomical locations. In the present examples, attempts were made to explore the regulatory roles of macrophages during early joint development; to investigate the regulatory links between macrophages and apoptotic cells, cartilage precursor cells / stem cells, and cartilage development; to clarify the spatiotemporal distribution and lineage origins of macrophages during joint development; and to investigate the impact of efferocytotic macrophages on cartilage regeneration in an adult osteoarthritis model.

[0022] Macrophages, as key immune cells within joints, not only regulate inflammation during the progression of osteoarthritis but also serve as a significant source of related inflammatory factors. Efferocytosis describes how phagocytes, particularly macrophages, rapidly and effectively eliminate apoptotic cells (ACs). This crucial process is crucial for biological development, immune regulation, and the maintenance of tissue homeostasis. Effective macrophage efferocytosis is followed by a resolution response, involving the secretion of various mediators, including TGF-β and interleukin-10 (IL-10), to suppress inflammation and promote tissue repair. The efferocytosis-induced macrophage inflammation resolution pathway is triggered by the activation of molecules produced by AC receptors or lysosomal degradation of internalized ACs. The present invention demonstrates how to induce macrophage efferocytosis by culturing macrophages with apoptotic cells in vitro, thereby achieving the highly effective promotion of cartilage regeneration in osteoarthritis by intra-articular injection of macrophages, thereby alleviating and treating osteoarthritis.

[0023] Example 1 In vitro model experiment In this example, an in vitro model was constructed to investigate the regulatory effects of macrophage efferocytosis on chondrogenic potential cells (MSCs and ADTC5 cells). Combined with in vivo single-cell transcriptome sequencing, the authors analyzed the regulation of MSCs and ADTC5 cells by signaling molecules potentially mediated by macrophage efferocytosis. The regulatory effects of the selected genes were validated by silencing them in an in vitro co-culture model. Furthermore, macrophages were induced with varying ratios of apoptotic cells (ACs) to determine the differential regulation of macrophages on chondrocyte differentiation and matrix secretion under different efferocytosis conditions. These results provide a deeper understanding of the in vitro regulation of macrophage efferocytosis during development and provide theoretical guidance for cartilage regeneration therapy.

[0024] Experimental methods: Bone Marrow Stromal Cells (BMSCs) extraction and culture experimental process: 1. Preparation of proliferation medium: Add fetal bovine serum (FBS) and penicillin-streptomycin solution (PS) to DMEM / F12 medium to a volume concentration of 10% FBS and 1% PS, respectively.

[0025] 2. Extraction of primary bone marrow cells: After sacrificing eight-week-old adult mice, soak them in 75% ethanol for 20 minutes and transfer them to a clean bench. Remove the skin and muscle tissue from the mouse legs and separate the entire hind limb bones, ensuring that the knee joint cavity is not exposed. Use surgical scissors to cut open the ends of the tibia and femur bone marrow cavity, rinse the bone marrow with PBS, and collect it in a 15mL centrifuge tube. Centrifuge the collected bone marrow at 1,000 rpm for 5 minutes, discard the supernatant, resuspend in the prepared cell proliferation medium, and inoculate it into a 10cm diameter cell culture dish. Culture in a cell culture incubator at 37°C and 5% CO2.

[0026] 3. Isolation of Primary Bone Marrow Cells: After approximately 8-12 hours of culture, the mesenchymal stem cells adhere to the cell wall and the medium is changed. Culture is continued in a cell culture incubator at 37°C and 5% CO2.

[0027] 4. Cell culture medium replacement: Change the culture medium every three days. Culture the cells in a cell culture incubator at 37°C and 5% CO2.

[0028] 5. Cell passaging: Cell passage is performed when the cell fusion reaches about 80%. After washing the culture dish with 5mL PBS, add 2mL 0.25% trypsin and digest in the incubator for 2 minutes. Gently tap the culture dish and observe that all the cells are floating and not attached to the wall. Then add 4mL proliferation medium to stop digestion and collect the liquid in a centrifuge tube. Centrifuge the collected cell suspension at 1000rpm for 5 minutes and discard the supernatant. Resuspend with cell proliferation medium and inoculate in a 10cm culture dish so that the cell inoculation density in the dish is 5000 / cm 2 .

[0029] 6. Cryopreservation: Digest and harvest the cells as described above, centrifuge at 1000 rpm for 5 minutes, and discard the supernatant. Resuspend in cell freezing buffer and transfer to cryovials. Store in a programmed cooling box at -80°C overnight, then freeze in liquid nitrogen.

[0030] 7. Cell recovery: Transfer the frozen cells in liquid nitrogen to a 37°C water bath and rewarm until completely thawed. Transfer the cell suspension to a centrifuge tube and centrifuge at 1000 rpm for 5 minutes. Discard the supernatant, resuspend in cell proliferation medium, and inoculate in a 10 cm culture dish to a cell seeding density of 5000 cells / cm 2 .

[0031] Bone Marrow-Derived Macrophages (BMDM) extraction and culture experimental process: 1. Preparation of Macrophage Differentiation Medium: Add fetal bovine serum (FBS) and penicillin-streptomycin solution (PS) to α-MEM medium to a final concentration of 10% FBS and 1% PS. Add macrophage colony-stimulating factor (M-CSF) to a final concentration of 50 ng / mL.

[0032] 2. Primary bone marrow cell extraction: The same experimental process as bone marrow mesenchymal stem cell extraction and culture.

[0033] 3. Isolation of Primary Bone Marrow Cells: After approximately 8-12 hours of culture, when the mesenchymal stem cells have adhered to the wall, remove any unattached cells and transfer them to a standard culture dish. Add 10 mL of macrophage differentiation medium and culture in a cell culture incubator at 37°C and 5% CO2.

[0034] 4. Macrophage differentiation culture: After 7 days of continuous culture, the mononuclear progenitor cells will differentiate into macrophages and can be used for experiments. Carefully scrape the macrophages with a cell scraper and set aside.

[0035] Adtc5 cell (chondrocyte) culture experimental process: 1. Preparation of proliferation medium: Add fetal bovine serum (FBS) and penicillin-streptomycin to DMEM / F12 medium. Then add FBS solution (PS) to make the volume concentration of FBS and PS 10% and 1%, respectively.

[0036] 2. Cell culture medium replacement: Same as the experimental process for bone marrow mesenchymal stem cell extraction and culture.

[0037] 3. Cell passage: Same as the experimental process for bone marrow mesenchymal stem cell extraction and culture.

[0038] 4. Cell recovery: Same as the experimental process for bone marrow mesenchymal stem cell extraction and culture.

[0039] Jurkat cell culture experimental process 1. Preparation of proliferation medium: Add fetal bovine serum (FBS) and penicillin-streptomycin solution (PS) to RPMI1640 medium to a volume concentration of 10% FBS and 1% PS, respectively.

[0040] 2. Cell culture medium replacement: Place the T75 culture flask upright and let it stand for 20 minutes. After the cells sink to the bottom of the flask, carefully aspirate an appropriate amount of culture medium from the supernatant. Then add an appropriate amount of fresh proliferation medium to the culture flask and gently pipette the cells to mix.

[0041] 3. Cell passaging: When the cell density exceeds 1×10 6 cells / mL, consider subculturing. Transfer the cell suspension in the flask to a centrifuge tube, centrifuge at 1200 rpm for 5 minutes, and discard the supernatant. Resuspend in cell proliferation medium and inoculate in a T75 culture flask to a cell seeding density of 1×10 5 cells / mL.

[0042] 4. Cell Cryopreservation: Transfer the cell suspension from the flask to a centrifuge tube and centrifuge at 1200 rpm for 5 minutes. Discard the supernatant. Resuspend in freezing buffer and transfer to a cryovial. Store in a programmed cooling box at -80°C overnight, then freeze in liquid nitrogen.

[0043] Macrophage burial co-culture model experimental process: 1. Ultraviolet light induces cell apoptosis: UV irradiation: Transfer the Jurkat cell suspension from the flask to a centrifuge tube and centrifuge at 1200 rpm for 5 minutes. Discard the supernatant. Resuspend in PBS and plate in a culture dish at a density of 1 × 105 cells / mL. Irradiate at 254 nm for 15 minutes. Then incubate in PBS for 2–3 hours before use in subsequent experiments.

[0044] 2. Cell Co-culture MSCs / adtc5 seeding: Add 2.6 mL of DMEM / F12 medium containing 10% FBS and 1% PS to a 6-well plate, and seed the digested MSCs or adtc5 at 5,000 cells / cm2 in the 6-well plate.

[0045] A Transwell chamber with a pore size of 0.4 µm was placed in the chamber. 1.5 mL of DMEM / F12 medium containing 10% FBS and 1% PS was added to the chamber. Mature differentiated macrophages were seeded at 5,000 cells / cm2 in the upper chamber, and Jurkat cells induced by ultraviolet light with apoptosis were seeded in the upper chamber according to the corresponding ratio (unless otherwise specified in the text, macrophages and ACs were seeded at the default ratio of 1:5).

[0046] Cell culture: Culture in a cell culture incubator at 37°C, 5% CO2 for 1 or 3 days.

[0047] Experimental results: 1. Effects of apoptotic cell-induced macrophages on mesenchymal stem cells and ADTC5 cells: Since a large number of macrophages and a certain number of apoptotic cells (ACs) are present during mouse knee joint development, it is speculated that macrophages are likely involved in the clearance of ACs, a process known as efferocytosis. In adults, macrophage efferocytosis contributes to inflammation resolution by releasing anti-inflammatory factors. However, whether macrophages also mediate the release of related factors during joint development, thereby regulating cartilage development, through efferocytosis remains unknown. To further explore the regulatory effects of macrophage efferocytosis on prechondrocytes and mesenchymal cells during development, the present invention constructed the aforementioned cell co-culture model for in vitro experimental investigation. Macrophages and ACs were seeded in the upper chamber of the co-culture to mimic the distribution of macrophages and ACs in the central region of the developing joint, with a default ratio of 1:5. MSCs or ADTC5 chondrogenic cells were seeded in the lower chamber of the co-culture to mimic the cells on the surface of developing articular cartilage. This allows for the investigation of the impact of macrophage efferocytosis on chondrogenesis.

[0048] First, the effect of UV-induced ACs in Jurkat cells was determined. Flow cytometry analysis of the induced Jurkat cells was performed using a gating strategy. PI and annexin staining allowed differentiation of cells at different stages of apoptosis. One hour after UV induction, Jurkat cells exhibited a clear trend toward apoptosis, with early apoptotic cells (PI-Annexin V-FITC+) accounting for 37.00 ± 0.25% and late apoptotic or necrotic cells (PI+Annexin V-FITC+) accounting for 36.40 ± 0.40%. Two hours after UV induction, a significant number of cells, accounting for 73.80 ± 0.43%, had entered a late apoptotic or necrotic state. Only a small number of cells, accounting for 3.62 ± 0.18% and 19.83 ± 0.33%, remained, either in the early apoptotic stage or in the early apoptotic stage, respectively. Six hours after UV induction, 81.87 ± 0.61% of the cells were in a late apoptotic or necrotic state. This suggests that a significant number of ACs can be obtained by incubating Jurkat cells for 2 hours after UV-induced apoptosis. Therefore, in subsequent co-culture models, ACs were obtained by incubating Jurkat cells for 2 hours after UV-induced apoptosis.

[0049] To further exclude the influence of macrophages and apoptotic cells in the coculture model on cell viability in the lower chamber, the viability of ADTC5 cells was measured on days 1, 3, and 6 of the coculture model. The results showed that on days 1 and 3, the cell viability of ADTC5 cells cocultured with macrophages and apoptotic cells in the upper chamber (MΦ+AC+) was not significantly different from that of the MΦ-AC-, MΦ+AC-, and MΦ-AC+ groups. Only on day 6 did the cell viability of the MΦ+AC+ group increase compared with the MΦ-AC- and MΦ-AC+ groups, but the increase was limited (1.037±0.009-fold and 1.254±0.011-fold, respectively). This result suggests that the presence of macrophages and apoptotic cells in the upper chamber of the coculture model has limited influence on the viability of cells in the lower chamber.

[0050] SOX9 is a key transcription factor in chondrocytes and is essential for developmental events including cartilage formation. Differentiated chondrocytes in articular hyaline cartilage synthesize more type II collagen and avoid the synthesis of type I collagen, resulting in cartilage degradation and fibrosis. In addition, Acan is an important gene encoding aggrecan in cartilage, which plays a key role in the morphogenesis of cartilage and bone. In order to examine whether the efferocytosis process of macrophages affects chondrogenic differentiation and cartilage matrix secretion, the gene expression levels of Sox9, Col1, Col2 and Acan were detected. For MSCs, after 1 day and 3 days of co-culture, the expression of Sox9, Col2 and Acan in MSCs co-cultured with macrophages and apoptotic cells (MΦ+AC+) in the upper chamber was increased compared with other groups (such as Figure 1 As shown in (A) and (B), Figure 1 The expression of chondrogenesis-related genes in the co-culture model is shown; (A) shows the gene expression level of MSCs after 1 day of co-culture, (B) shows the gene expression level of MSCs after 3 days of co-culture, (C) shows the gene expression level of adtc5 cells after 1 day of co-culture, and (D) shows the gene expression level of adtc5 cells after 3 days of co-culture). Specifically, the expression of Sox9 in the MΦ+AC+ group was upregulated by 2.87±0.16 times (P<0.0001), 2.70±0.15 times (P<0.0001), and 2.49±0.14 times (P<0.0001) compared with the MΦ-AC-, MΦ+AC-, and MΦ-AC+ groups on day 1, and by 1.90±0.06 times (P<0.0001) on day 3. .0001), 1.95±0.06-fold (P<0.0001), and 2.28±0.07-fold (P<0.0001); the expression of Col2 in the MΦ+AC+ group was upregulated by 12.01±1.05-fold (P<0.0001), 7.55±0.66-fold (P<0.0001), and 1.95±0.06-fold (P<0.0001), respectively, compared with the MΦ-AC-, MΦ+AC-, and MΦ-AC+ groups on the first day. ) and 11.92±1.04 times (P<0.0001), and were up-regulated by 5.89±0.67 times (P=0.0001), 4.09±0.46 times (P=0.0003) and 3.28±0.37 times (P=0.0005) on the third day, respectively; the expression of Acan in the MΦ+AC+ group was significantly higher than that in the MΦ-AC-, MΦ+AC- and MΦ-AC+ groups on the first day. They were upregulated by 4.32±0.13-fold (P<0.0001), 2.02±0.06-fold (P<0.0001), and 3.52±0.11-fold (P<0.0001), respectively, and by 2.59±0.28-fold (P=0.0005), 2.65±0.29-fold (P=0.0005), and 2.72±0.30-fold (P=0.0004), respectively, on the third day (e.g. Figure 1After 1 and 3 days of co-culture, the expression of Col1 in MSCs co-cultured with MΦ+AC+ was not different from that in other groups.

[0051] For adtc5 cells, after one day of co-culture, the expression of Sox9 in adtc5 cells co-cultured with MΦ+AC+ was increased compared with other groups, and was upregulated by 3.19±0.12 times (P<0.0001), 1.74±0.06 times (P<0.0001), and 2.77±0.10 times (P<0.0001) compared with the MΦ-AC-, MΦ+AC-, and MΦ-AC+ groups, respectively (e.g. Figure 1 The expression of Col1 and Col2 was not significantly different from that of the other groups. The expression of Acan in adtc5 cells co-cultured with MΦ+AC+ was increased by 5.62±0.10 times (P=0.0001) and 3.40±0.06 times (P=0.0003) compared with the MΦ-AC- and MΦ-AC+ groups, but was not significantly different from the MΦ+AC- group (P=0.4125) (as shown in Figure 2). Figure 1 After 3 days of co-culture, the expressions of Sox9, Col1, Col2, and Acan in adtc5 cells co-cultured with MΦ+AC+ were increased compared with those in other groups (as shown in Figure 2). Figure 1 Specifically, the expression of Sox9 in the MΦ+AC+ group was upregulated by 2.60±0.17-fold (P<0.0001), 2.31±0.15-fold (P<0.0001), and 2.19±0.14-fold (P<0.0001) compared with the MΦ-AC-, MΦ+AC-, and MΦ-AC+ groups, respectively. The expression of Col1 in the MΦ+AC+ group was upregulated by 2.00±0.13-fold (P=0.0016), 1.68±0.11-fold (P=0.0063), and 2.12±0.14-fold (P=0.0011) compared with the MΦ-AC-, MΦ+AC-, and MΦ-AC+ groups, respectively. The expression of Col2 was upregulated by 2.16±0.11-fold (P=0.0006), 1.84±0.09-fold (P=0.0017), and 2.45±0.12-fold (P=0.0003) in the MΦ+AC+ group compared with the MΦ-AC-, MΦ+AC-, and MΦ-AC+ groups, respectively. The expression of Acan was upregulated by 1.66±0.14-fold (P=0.0165), 1.71±0.14-fold (P=0.0131), and 1.81±0.15-fold (P=0.0086) in the MΦ+AC+ group compared with the MΦ-AC-, MΦ+AC-, and MΦ-AC+ groups, respectively (e.g. Figure 1 (shown in (D)).

[0052] Overall, in the co-culture environment of ACs-induced macrophages, the expression levels of cartilage-related genes in both MSCs and ADTC5 cells indicated a stronger chondrogenic capacity. Furthermore, Col2 expression was consistently high in both MSCs and ADTC5 cells, while Col1 expression was not significantly elevated in MSCs, demonstrating the regulatory tendency of ACs-induced macrophages towards hyaline cartilage in this environment.

[0053] 2. Effects of inhibiting macrophage efferocytosis on mesenchymal stem cells and ADTC5 cells: To verify whether ACs induce macrophage efferocytosis in the co-culture model, expression of efferocytosis-related genes was examined. Typically, efferocytosis upregulates the downstream COX2 / PGE2 pathway, with COX2 being encoded by Ptgs2. Furthermore, efferocytosis-mediated Ptgs2 induction requires the DNA methyltransferase DNMT3A, which functions during late embryonic and postnatal life. To distinguish mRNA expression between mouse BMDM and apoptotic human Jurkat cells, mouse-specific primers were designed. After 1 hour of co-culture of BMDM with apoptotic Jurkat cells, expression of Ptgs2 and Dnmt3a was upregulated by 11.36±0.46-fold (P<0.0001) and 1.28±0.03-fold (P=0.0027), respectively. To verify whether this upregulation was altered by attenuation of efferocytosis, BMDM from Cx3cr1GFP / GFP mice, whose cells express green fluorescent protein (GFP) instead of CX3CR1, were subjected to the same treatment as described below. Figure 2 Cx3cr1 - / -CX3CR1 is a key "find me" signaling receptor in macrophage clearance of ACs. CX3CR1 deficiency reduces the efficiency of macrophage clearance of ACs. After 1 hour of incubation of CX3CR1-deficient BMDM with apoptotic Jurkat cells, the expression of Ptgs2 and Dnmt3a was significantly suppressed compared to BMDM from wild-type mice (P<0.0001, P<0.0001), indicating that efferocytosis is reduced in CX3CR1-deficient BMDM. Furthermore, Lrg1 and Tgf-β1 expression were also assessed. After 6 hours of incubation of BMDM with apoptotic Jurkat cells, both Lrg1 and Tgf-β1 expression were upregulated, by 1.43±0.04-fold and 1.48±0.06-fold, respectively (P=0.0020, P=0.0099). However, no upregulation was observed in CX3CR1-deficient BMDM, likely due to the inhibition of efferocytosis. The same trend was also verified by detecting macrophage LRG1 and TGF-β1 at the protein level, that is, the expression of LRG1 and TGF-β1 in macrophages lacking CX3CR1 was inhibited after ACs induction.

[0054] BafilomycinA1 is a lysosomal vacuolar H+-ATPase inhibitor that blocks the degradation of ACs by phagolysosomes during efferocytosis. Macrophages pretreated with bafilomycin also showed that the expression of Lrg1 and Tgf-β1, which should have been upregulated, was suppressed in response to ACs, with no significant upregulation. This demonstrates that AC-induced upregulation of Lrg1 and Tgf-β1 in BMDMs is mediated by efferocytosis.

[0055] To further examine the effect of macrophage efferocytosis on the chondrogenic differentiation of BMSCs and adtc5 cells, BMDM lacking CX3CR1 (Cx3cr1) were used in the co-culture system. - / - For MSCs and adtc5 cells, Cx3cr1 - / - In the group with or without ACs, the expression of cartilage-related genes in BMSCs and adtc5 was no longer significantly upregulated compared with the WT group (e.g. Figure 2 As shown, Figure 2 Figure 3 shows the effect of CX3CR1 deficiency on chondrogenesis-related gene expression in BMDMs in the co-culture model; (A) shows the gene expression level of MSCs after 1 day of co-culture, (B) shows the gene expression level of MSCs after 3 days of co-culture, (C) shows the gene expression level of adtc5 cells after 1 day of co-culture, and (D) shows the gene expression level of adtc5 cells after 3 days of co-culture).

[0056] These results indicate that culturing BMDM with ACs induces BMDM efferocytosis. When BMDM efferocytosis is inhibited, ACs no longer upregulate BMDM expression of Lrg1 and Tgf-β1. Furthermore, ACs also abolish the BMDM-induced chondrogenic differentiation and cartilage matrix secretion of MSCs and adtc5 cells. It is further hypothesized that BMDM efferocytosis regulates chondrogenesis through the paracrine effects of LRG1 and TGF-β1, and that inhibition of efferocytosis suppresses this paracrine regulation.

[0057] Therefore, it can be proved that ACs-induced macrophages can significantly enhance the cartilage matrix secretion ability of MSCs and adtc5 cells.

[0058] 3. Effect of the number of apoptotic cells on the expression of LRG1 and TGF-β1 in macrophages: To investigate whether the difference in macrophage efferocytosis levels would affect the expression of Lrg1 and Tgf-β1 and further affect the chondrogenesis of MSCs and adtc5 cells, BMDM and ACs in different ratios were cultured. The results showed that the expression of Lrg1 in BMDM increased with the increase of ACs ratio (e.g. Figure 3 As shown in part (A), Figure 3 The effects of different ratios of BMDM and ACs on the expression of Lrg1 and Tgf-β1 are shown; (A) shows the gene expression level of Lrg1 after BMDM was incubated with apoptosis-inducing Jurkat for 6 hours, and (B) shows the gene expression level of Tgf-β1 after BMDM was incubated with apoptosis-inducing Jurkat for 6 hours. ). Tgf-β1 expression was highest when the ratio of BMDM to ACs was 1:5. Compared with the ratios of 1:2 and 1:10, the expression levels were increased by 1.36±0.06 times (P=0.0022) and 1.39±0.06 times (P=0.0014), respectively (as shown in Figure 2). Figure 3 (shown in part (B)).

[0059] LAMP1 is a late phagosome and lysosome marker that accumulates in phagosomes during efferocytosis. Immunofluorescence staining of BMDM revealed that after one day of culture, the fluorescence intensity of LAMP1 and LRG1 in BMDM increased with the increase in the ratio of ACs; while the fluorescence intensity of TGF-β1 was highest when the ratio of BMDM to ACs was 1:5. Compared with the ratios of 1:0, 1:2, and 1:10, the fluorescence intensity increased by 3.10±0.15 times (P<0.0001), 4.17±0.20 times (P<0.0001), and 4.90± The fluorescence intensity of LAMP1 in BMDM increased by 0.23 times (P<0.0001) after 3 days of culture with the increase of ACs ratio; the fluorescence intensity of TGF-β1 was the highest when the ratio of BMDM to ACs was 1:5, and compared with the ratios of 1:0 and 1:2, the fluorescence intensity was increased by 1.51±0.16 times (P=0.0256) and 1.53±0.16 times (P=0.0220), respectively; while the fluorescence intensity of LRG1 was no different from that of the other groups.

[0060] Immunofluorescence results on day 1 showed that BMDM and different ACs exhibited increased efferocytosis with increasing AC number, and the trends for LRG1 and TGF-β1 were consistent with the mRNA expression results. Immunofluorescence results on day 3 showed that the TGF-β1 trend in BMDM continued, while the LRG1 trend subsided. In summary, macrophage synthesis and secretion of LRG1 increased with increasing efferocytosis levels, while TGF-β1 showed an AC number-dependent trend of initially increasing and then decreasing, with high levels of TGF-β1 being synthesized and secreted only at appropriate efferocytosis levels.

[0061] To further examine whether the differences in LRG1 and TGF-β1 synthesis and secretion caused by the level of efferocytosis would lead to different regulation of cell chondrogenesis, BMDM and ACs were seeded at 1:0, 1:2, 1:5, and 1:10 in the co-culture model. For MSCs, after 1 and 3 days of co-culture, the expression of Sox9, Col2, and Acan in MSCs in the BMDM and ACs ratio group of 1:5 was significantly upregulated compared with those in the 1:0, 1:2, and 1:10 groups (e.g. Figure 4 As shown in (A) and (B), Figure 4Figure 2 shows the effect of different ratios of BMDM and ACs in the co-culture model on the expression of chondrogenesis-related genes, where (A) shows the gene expression levels of MSCs after 1 day of co-culture, and (B) shows the gene expression levels of MSCs after 3 days of co-culture). Specifically, the expression of Sox9 in the 1:5 group was increased by 2.69±0.31-fold (P=0.0005), 1.87±0.22-fold (P=0.0036), and 1.75±0.20-fold (P=0.0060) on day 1, and by 1.98±0.07-fold (P<0.0001), 1.42±0.05-fold (P=0.0027), and 1.47±0.05-fold (P=0.0017) on day 3 compared with the 1:0, 1:2, and 1:10 groups. The expression of Col2 in the 1:5 group was increased by 7.80±0.42-fold (P<0.0001), 3.11±0.17-fold (P<0.0001), and 3.31±0.20-fold (P<0.0001) on day 1 compared with the 1:0, 1:2, and 1:10 groups, respectively. The expression of Acan in the 1:5 group was upregulated by 2.02±0.17-fold (P=0.0004), 1.47±0.12-fold (P=0.0077) and 1.62±0.14-fold (P=0.0026) compared with the 1:0, 1:2 and 1:10 groups on the first day, and by 2.65±0.11-fold (P<0.0001), 2.38±0.10-fold (P<0.0001) and 2.22±0.09-fold (P<0.0001) on the third day, respectively. As for the expression of Col1, on day 3, the 1:5 group was significantly downregulated by 0.40±0.06 times compared with the 1:10 group (P=0.0023) (e.g. Figure 4 (A)). This indicates that different ratios of BMDM to ACs produce varying chondrogenic effects on MSCs. A 1:5 BMDM to AC ratio promoted chondrogenic differentiation and matrix expression in MSCs, both in the short term (1 day) and the long term (3 days). A 1:10 BMDM to AC ratio abolished this facilitating effect, and even resulted in increased Col1 expression in MSCs over the long term (3 days). This suggests that appropriate efferocytosis is required for macrophages to regulate chondrogenesis; when macrophages are required to clear more ACs, chondrogenic activity is suppressed.

[0062] In summary, macrophage synthesis and secretion of LRG1 increased with increasing levels of efferocytosis, whereas macrophage synthesis and secretion of TGF-β1 initially increased and then decreased with increasing levels of efferocytosis. Macrophages' promotion of chondrogenesis in MSCs and ADTC5 cells requires the presence of an appropriate number of ACs. It is hypothesized that macrophage efferocytosis regulates chondrogenic differentiation through two key signaling axes: the pro-chondrogenic axis (TGF-β1), whereby an appropriate number of ACs induces macrophage secretion of TGF-β1, driving the expression of Sox9, Col2, and Acan in MSCs and ADTC5 cells. The anti-chondrogenic axis (LRG1), whereby an increase in the number of ACs increases macrophage LRG1 secretion, inhibits chondrogenesis. In an in vitro co-culture model, the optimal ratio of macrophages to ACs for chondrogenesis is 1:5.

[0063] In this study, researchers established an in vitro co-culture model to investigate the regulatory mechanisms of AC-induced macrophage efferocytosis on MSCs and adtc5 chondrogenic cells. The results revealed a molecular network by which macrophages dynamically balance cartilage differentiation and matrix synthesis through the secretion of LRG1 and TGF-β1. The study found that after AC clearance (efferocytosis), macrophages significantly upregulated chondrogenesis-related genes (such as Sox9, Col2, and Acan) while suppressing the expression of the fibrosis marker Col1, thereby promoting hyaline cartilage formation. This regulatory effect was strictly conditional: chondrogenesis was activated only when macrophages were co-cultured with ACs (MΦ+AC+ group), but not when macrophages were co-cultured with either macrophages (MΦ+AC- group) or apoptotic cells (MΦ-AC+ group). Genetic knockout (Cx3cr1GFP / GFP) and pharmacological inhibition (BafilomycinA1) confirmed that CX3CR1-dependent efferocytosis is a core component in regulating LRG1 / TGF-β1 signaling. Lack of CX3CR1 or blocking lysosomal degradation both resulted in a weakened chondrogenic effect, suggesting that the integrity of efferocytosis is a necessary condition for signal transduction. Mechanistically, efferocytosis regulates cartilage differentiation through two key pathways: 1. Chondrogenic signaling axis (TGF-β1): ACs induce macrophages to secrete TGF-β1, which activates the SMAD2 / 3 pathway in MSCs and adtc5 cells, driving the expression of Sox9, Col2, and Acan. TGF-β1 secretion is dose-dependent, reaching a peak at a macrophage to AC ratio of 1:5, at which point the chondrogenic effect is strongest.

[0064] 2. Chondrogenesis-inhibiting signaling axis (LRG1): Effeminate phagocytosis also induces macrophage secretion of LRG1. Silencing LRG1 further amplifies the chondrogenic effect, suggesting that LRG1 acts as a "brake" signal in effeminate phagocytosis, preventing excessive and ectopic chondrogenesis.

[0065] The study further revealed that efferocytosis exerts a dual regulatory effect on macrophage phenotype. Efferocytosis maintains a dynamic balance, with the number of apoptotic cells determining the direction of regulation. A low ratio of ACs (1:2) is insufficient to activate TGF-β1 signaling, while a high ratio (1:10) may inhibit chondrogenesis through excessive LRG1 production. Only a 1:5 ratio achieves optimal chondrogenic effects.

[0066] The present invention demonstrates the dual role of macrophage efferocytosis in cartilage development. On the one hand, it promotes chondrogenic differentiation through TGF-β1, and on the other hand, it restricts excessive proliferation through LRG1, forming a precise regulatory network. This discovery provides a new perspective for understanding immune-mesenchymal interactions in joint development and provides a theoretical basis for cartilage repair strategies, such as the design of biomaterials based on efferocytosis.

[0067] Example 2 In vivo model experiment In the examples of this chapter, we will first determine the efferocytosis level of macrophages at different locations by performing more detailed zoning of the knee joint of developing mice. We will then examine the expression of TGF-β1 and LRG1 in macrophages at different locations and correlate this with the efferocytosis level to verify whether the differences in TGF-β1 and LRG1 expression in macrophages at different efferocytosis levels at different locations within the joint during development are consistent with the in vitro results. GFP / GFP Finally, the researchers used embryonic macrophages and ACs-induced macrophages to treat osteoarthritis, a disease characterized by large cartilage defects, to verify that embryonic knee macrophages and ACs-induced macrophages have excellent cartilage regeneration regulation and potential for disease treatment.

[0068] Experimental methods: The process of establishing the mouse osteoarthritis model: 1. Pre-experimental preparation: Dissolve sodium iodoacetate in PBS to a final concentration of 200 mg / mL and filter through a 0.2 µm filter. Anesthetize mice with 4% isoflurane in an air anesthesia machine. After securing them to the operating table, anesthesia was maintained with 1.5% isoflurane. Shave the hair on the right hind leg of the mouse with a razor and disinfect the skin with iodine.

[0069] 2. Intra-articular injection: Use a 20 μL microsyringe to inject the prepared sodium iodoacetate solution into the mouse knee joint cavity, injecting 10 μL per mouse.

[0070] 3. Subsequent treatment tests can be carried out after the mice have been raised for two weeks.

[0071] Treatment process of mouse osteoarthritis model: Mouse adult BMDM extraction and culture process: 1. Extraction and culture: Same as the extraction and culture process of bone marrow-derived macrophages.

[0072] 2. Acquisition of apoptotic cells: the same process as ultraviolet-induced cell apoptosis.

[0073] 3. Induction of BMDM with Apoptotic Cells: Add apoptotic cells at the predetermined ratio to the BMDM culture dish in differentiation culture. Incubate at 37°C for 6 hours, then discard the supernatant. Wash once with PBS and digest with 0.25% trypsin at 37°C for 2 minutes. Gently tap the dish to observe that all cells are floating and not attached. Add 4 mL of proliferation medium to terminate the digestion and collect the liquid in a centrifuge tube. Centrifuge the collected cell suspension at 1,000 rpm for 5 minutes, discard the supernatant, add PBS, and set aside.

[0074] Cell therapy process for mouse osteoarthritis model: 1. Experimental Preparation: Pre-treatment macrophages were dispersed in PBS at a concentration of 1 × 10⁶ cells / mL. Mice were anesthetized with 4% isoflurane in an air anesthesia machine and then secured to the operating table under continuous anesthesia with 1.5% isoflurane. Hair on the right hind leg of the mouse was shaved with a razor, and the skin surface was disinfected with iodine.

[0075] 2. Intra-articular Injection: Use a 20 μL microsyringe to inject the prepared cell suspension into the mouse knee joint cavity. Inject 5 μL per mouse, containing 5,000 cells. Inject twice a week for 3 weeks.

[0076] Experimental results: 1. Spatial and temporal distribution of LRG1 and TGF-β1 secretion by macrophages In the present embodiment, validating the impact of macrophage efferocytosis on mouse articular cartilage development during development requires more detailed regional delineation. Sections at the peripheral (meniscus) and central (cruciate ligament) locations can be further divided into two regions: the center (away from the cartilage surface) and the edge (close to the cartilage surface) based on the number of apoptotic cells surrounding macrophages. Thus, sections of mouse joints during development can be divided into: the center (away from the cartilage surface) of the central (cruciate ligament) section, the edge (close to the cartilage surface) of the central (cruciate ligament) section, the center (away from the cartilage surface) of the peripheral (meniscus) section, and the edge (close to the cartilage surface) of the peripheral (meniscus) section.

[0077] First, it was necessary to determine the level of efferocytosis of macrophages at different locations during mouse knee joint development, specifically the number of ACs engulfed by macrophages. LAMP1, a late phagosome and lysosome marker, accumulates in phagosomes undergoing efferocytosis. Immunofluorescence staining revealed that at E16.5, LAMP1 fluorescence intensity in macrophages located at the center of central sections was significantly higher than that in macrophages located at the edge of central sections and at the center of peripheral sections (P=0.0007, P=0.0029, respectively). However, no significant differences in LAMP1 fluorescence intensity were observed between macrophages located at the edge of central sections, the center of peripheral sections, or the edge of peripheral sections.

[0078] At E18.5, LAMP1 fluorescence intensity in macrophages located in the center of central sections remained high and significantly different from that in macrophages located at the edge of central sections and the center of peripheral sections (P=0.0219, P=0.0060, respectively). However, no significant differences were observed between macrophages located at the edge of central sections, the center of peripheral sections, or the edge of peripheral sections. At P1, LAMP1 fluorescence intensity in macrophages located in the center of central sections no longer differed significantly from that in macrophages located at the edge of central sections or the center of peripheral sections. However, LAMP1 fluorescence intensity in macrophages located at the edge of central sections was significantly higher than that in macrophages located at the edge of peripheral sections (P=0.0443). LAMP1 fluorescence staining results indicate that during mouse joint development, macrophages located in different locations phagocytose ACs to varying degrees, indicating that the level of efferocytosis varies. Overall, macrophage efferocytosis was strong in the most central location of the joint (center of central slices) between E16.5 and E18.5, exceeding that of macrophages in other locations. By P1, macrophage efferocytosis in the most central location of the joint decreased and remained unchanged from that in other regions, likely due to the substantial clearance of ACs. Furthermore, macrophage efferocytosis was only high in the center of central slices, with little difference in the center and edges of peripheral slices. This may be due to the fact that, from a three-dimensional perspective, peripheral slices are generally located away from the center of the joint and away from locations where ACs are more numerous.

[0079] These results suggest that during development, macrophages in mouse joints phagocytose ACs at different levels at different locations. Macrophages exhibit higher levels of efferocytosis in the center of the joint, away from the cartilage surface, and lower levels closer to the cartilage surface.

[0080] Immunofluorescence staining revealed that at E16.5, the TGF-β1 fluorescence intensity of macrophages located at the edge of central sections was significantly higher than that of macrophages located at the center and edge of peripheral sections (P<0.0001, P=0.0011). However, there was no significant difference in TGF-β1 fluorescence intensity between macrophages located at the center, center, or edge of peripheral sections. At E18.5, the TGF-β1 fluorescence intensity of macrophages located at the edge of central sections remained high, significantly different from that of macrophages located at the center and edge of peripheral sections (P=0.0024, P=0.0113). However, there was no significant difference in TGF-β1 fluorescence intensity between macrophages located at the center, center, or edge of peripheral sections. At P1, the TGF-β1 fluorescence intensity of macrophages at the edge of the central slice was still significantly different from that of macrophages at the center of the central slice (P=0.0352), but was not significantly different from that of macrophages at the edge of the peripheral slice. In addition, the TGF-β1 fluorescence intensity of macrophages at the edge of the peripheral slice was significantly stronger than that of macrophages at the center of the peripheral slice (P<0.0001). Overall, at E16.5-P1, TGF-β1 expression was higher in macrophages near the cartilage in the center of the joint (at the edge of the central slice), higher than in macrophages at other locations. When mice are at P1 after birth, the expression of TGF-β1 in macrophages near the cartilage at the periphery of the joint (at the edge of the peripheral slice) is also higher than that in macrophages far away from the cartilage. These results indicate that during joint development, macrophages close to the surface of cartilage formation secrete TGF-β1 to promote the chondrogenesis of cartilage progenitor cells / chondrocytes.

[0081] These results suggest that during development, macrophages in mouse joints express different levels of TGF-β1. Macrophages in joints close to the cartilage surface express higher levels of TGF-β1, while those in joints further from the cartilage surface express lower levels.

[0082] Immunofluorescence staining revealed that at E16.5, LRG1 fluorescence intensity was high in macrophages located in the center of central sections of joints, significantly higher than that in macrophages located at the edge of central sections and the center of peripheral sections (P=0.0059, P=0.0002). However, no significant differences in LRG1 fluorescence intensity were observed between macrophages located at the edge of central sections, the center of peripheral sections, or the edge of peripheral sections. At E18.5, LRG1 fluorescence intensity remained high in macrophages located at the edge of central sections, significantly different from that in the center of central sections (P=0.0067), but not at the center of peripheral sections. There was no significant difference in LRG1 fluorescence intensity between macrophages located at the center of central sections, the center of peripheral sections, or the edge of peripheral sections. At P1, no significant differences in LRG1 fluorescence intensity were observed among macrophages located at any specific location within the joint. Overall, LRG1 expression was higher in macrophages located in the most central part of the joint (center of the central slice) between E16.5 and E18.5, compared to macrophages located in several other locations. By postnatal day P1, LRG1 expression in macrophages at various locations within the joint no longer differed.

[0083] These results indicate that during development, macrophages in mouse joints express different levels of LRG1. Macrophages in joints close to the cartilage surface exhibit lower levels of LRG1, while those further from the cartilage surface exhibit higher levels.

[0084] 2. Regulatory effects of macrophage efferocytosis on articular cartilage regeneration The present invention establishes a mouse osteoarthritis (OA) model, and uses this large-area cartilage damage model for testing. After the onset of OA, treatment is performed by injecting cells into the joint cavity, twice a week, with 5,000 cells injected each time, for a total of 3 weeks (e.g. Figure 5 As shown in (A), Figure 5A schematic diagram shows how ACs-pretreated BMDM promote cartilage regeneration in osteoarthritis (OA). (A) shows the experimental schematic. Briefly, one week after the OA model was established, the corresponding cell injections were performed twice weekly for three weeks. Subsequently, mouse joints were harvested. (B) Shows representative images of safranin O- and fast green-stained knee joint sections after OA modeling, following continuous injection of PBS, BMDM, ACs-induced BMDM (1:5), and ACs-induced BMDM (1:10) for three weeks, followed by sham surgery. (Bar = 500 μm) (C) Shows a heatmap of the Mankin scoring system variables in (A). (D) Shows statistical analysis of cartilage destruction in each treatment group using the Mankin scoring system. (n = 6, mean ± SD) (E) Shows representative images of HE-strained knee joint sections after OA modeling, following continuous injection of PBS, BMDM, ACs-induced BMDM (1:5), and ACs-induced BMDM (1:10) for three weeks, followed by sham surgery. The black dashed circle outlines the synovium. bar=200μm, (F) shows the statistical analysis of synovial area in different groups, n=6, mean ± SD, (G) shows the joint sections of OA model were continuously injected with PBS, BMDM, ACs-induced BMDM (1:5) and ACs-induced BMDM (1:10) for 3 weeks, sham operation, and immunofluorescence staining was used to analyze the expression of COL2 in cells, COL2, green, bar=200μm, (H) shows the joint sections of OA model were continuously injected with PBS, BMDM, ACs-induced Joint sections of BMDM (1:5) and ACs-induced BMDM (1:10) were sliced for 3 weeks and sham treatment was performed. Immunofluorescence staining was used to analyze the expression of SOX9 in cells. SOX9, green, bar = 200 μm. (I)-(J) show the statistical analysis of the percentage of COL2+ cells and fluorescence intensity in the cartilage of different groups, n = 6, mean ± SD. (K)-(L) show the statistical analysis of the percentage of SOX9+ cells and fluorescence intensity in the cartilage of different groups, n = 6, mean ± SD).

[0085] In Example 1, an in vitro co-culture model revealed that macrophage efferocytosis, through TGF-β1 and LRG1, exerts both positive and negative regulatory effects on chondrogenesis. In particular, when macrophages and apoptotic cells were cultured in a 1:5 ratio, the best effect was achieved in promoting chondrogenic differentiation and cartilage matrix secretion. To this end, after establishing an osteoarthritis model, the therapeutic effect was verified by intra-articular injection of apoptotic BMDM. The experiment was divided into five groups: uninduced OA (Sham), OA-induced PBS-treated group (PBS), OA-induced BMDM-treated group (BMDM), OA-induced BMDM-treated group (ACs-induced BMDM (1:5), hereinafter referred to as the BMDM-ACs 1:5 group), and OA-induced BMDM-treated group (ACs-induced BMDM (1:10), hereinafter referred to as the BMDM-ACs 1:10 group).

[0086] The sliced tissue was stained with safranin fast green. The depth of safranin staining can reflect the content of proteoglycans in the cartilage. The results showed that the cartilage surface of the BMDM-ACs 1:5 group was continuous and smooth, with a deeper and uniform distribution of safranin staining, which was very close to that of the sham group. The cartilage surface of the BMDM-ACs 1:10 group was slightly less smooth, with a deeper but uneven distribution of safranin staining, and obvious damage (such as Figure 5 The regeneration effect of cartilage in the osteoarthritis model was evaluated by Mankin scoring based on the results of safranin fast green staining. A higher score indicates more severe cartilage damage. Based on cartilage structure, cellularity, proteoglycan consumption, and tide line integrity, it can be seen that, with the exception of the sham group, the BMDM-ACs 1:5 group had the best cartilage tissue regeneration effect (see Figure 2). Figure 5 (C)). According to the total score, except for the Sham group which scored close to 0, the BMDM-ACs1:5 group scored the lowest, 1.83 points, which was significantly different from the PBS, BMDM, and BMDM-ACs1:10 groups (P<0.0001, P=0.0023, P=0.0004). The BMDM group ranked second, followed by the BMDM-ACs1:10 group, which scored 4.17 points, which was significantly different from the PBS group (P<0.0001) but not different from the BMDM group (as shown in Figure 2). Figure 5 (D) ). This suggests that the BMDM-ACs 1:5 ratio significantly enhances cartilage regeneration in OA. However, the ACs-induced BMDM group (BMDM:ACs = 1:10) and the BMDM group (BMDM:ACs = 1:0) exhibited relatively weaker promoting effects. This confirms that macrophages can only effectively regulate and promote cartilage regeneration in OA under appropriate efferocytosis.

[0087] The sliced tissues were stained with HE to examine the level of synovial hypertrophy. The results showed that the synovial area in the BMDM-ACs 1:5 group and the BMDM-ACs 1:10 group was smaller, with no obvious hyperplasia, which was very close to that of the Sham group (e.g. Figure 5 The synovial areas of the BMDM-ACs1:5 group and BMDM-ACs1:10 group were 0.37±0.024 times (P<0.0001) and 0.51±0.028 times (P<0.0001) compared with the PBS group, respectively, demonstrating its good inhibitory effect on joint inflammation (e.g. Figure 5 (F)). However, there was no difference in synovial area between the BMDM-ACs1:5 group and the BMDM-ACs1:10 group (P=0.2200), indicating that there was no significant difference in the improvement of inflammation caused by macrophage treatment induced by different numbers of apoptotic cells, and both groups could effectively inhibit the progression of inflammation caused by joint damage. Immunofluorescence staining of joint sections of each group showed that after treatment, the COL2-positive ratio of chondrocytes in the BMDM-ACs1:5 group was significantly higher than that in the PBS group, BMDM group, and BMDM-ACs1:10 group, at 70.90±2.47%, which was close to the 73.67±2.38% of the Sham group (as shown in Figure 5). Figure 5 Meanwhile, the fluorescence intensity of COL2-positive chondrocytes in the BMDM-ACs1:5 group was also higher, which was 1.77±0.097 times (P<0.0001), 1.57±0.086 times (P=0.0006), and 1.52±0.083 times (P=0.0013) compared with those in the PBS group, BMDM group, and BMDM-ACs1:10 group, respectively (as shown in (G) and (I)). Figure 5 (J). However, there was no significant difference in the COL2-positive ratio and fluorescence intensity of chondrocytes between the PBS group and the BMDM1:10 group. This shows that the ACs-induced BMDM group (BMDM:ACs=1:5) has a promoting effect on the COL2 expression of chondrocytes, while the ACs-induced BMDM group (BMDM:ACs=1:10) has a limited promoting effect on the COL2 expression of chondrocytes. Compared with the PBS group, BMDM group and BMDM-ACs1:10 group, the SOX9-positive ratio of chondrocytes in the BMDM-ACs1:5 group was significantly higher, at 57.99±2.53% (as shown in Figure 2). Figure 5 At the same time, the fluorescence intensity of SOX9-positive chondrocytes in the BMDM-ACs1:5 group was also higher than that in the PBS group, which was 3.47±0.376 times that of the PBS group (P<0.0001) (as shown in (H) and (K)). Figure 5(L)). The SOX9-positive ratio and fluorescence intensity of chondrocytes in the 1:10 BMDM-ACs group were significantly higher than those in the PBS group, while the SOX9-positive ratio was lower than that in the 1:5 BMDM-ACs group. This suggests that the 1:5 BMDM-ACs ratio promotes SOX9 expression in chondrocytes. In summary, under appropriate efferocytosis, macrophages promote SOX9 and COL2 expression in chondrocytes of OA cartilage, alleviating the decreased SOX9 and COL2 expression in chondrocytes caused by OA, thereby further promoting cartilage regeneration. The optimal ratio for pre-induction of BMDMs with ACs is 1:5 BMDM:ACs.

[0088] In this example, mouse knee joints were divided into four regions: the center of the central section (away from the cartilage surface), the edge of the central section (close to the cartilage surface), the center of the peripheral section (away from the cartilage surface), and the edge of the peripheral section (close to the cartilage surface) to investigate the spatial specificity of macrophage efferocytosis during joint development. The study revealed a unique spatial division of labor among macrophages during joint development: in the central region, away from the cartilage surface, high levels of efferocytosis drive LRG1 expression, inhibiting cartilage differentiation and maintaining joint cavity morphology. In contrast, in the peripheral region, close to the cartilage surface, moderate efferocytosis induces TGF-β1 secretion, promoting chondrogenic differentiation and matrix deposition by chondrogenic progenitor cells. This bidirectional "central inhibition-peripheral promotion" regulatory network ensures the spatiotemporal precision of cartilage differentiation. Furthermore, using a CX3CR1-deficient mouse model, they found that transient loss of efferocytosis during early development leads to impaired cartilage matrix synthesis (reduced COL2 deposition and decreased proteoglycan content), highlighting the importance of efferocytosis within a critical time window.

[0089] Based on the above mechanism, the present invention proposes a therapeutic strategy targeting efferocytosis: by optimizing the ratio of macrophages to apoptotic cells (1:5) to simulate the developmental microenvironment, BMDM are reprogrammed to a regenerative phenotype, significantly improving the cartilage regeneration effect; deviation from this ratio (such as 1:10) results in poor regeneration effect, confirming that precise regulation of efferocytosis levels is the key to successful treatment.

[0090] Based on the above research, an embodiment of the present invention proposes a use of macrophages pretreated with apoptotic cells in the preparation of an osteoarthritis treatment preparation, wherein the macrophages pretreated with apoptotic cells are used to alleviate the inflammatory effects of osteoarthritis and promote osteoarthritis cartilage regeneration, including promoting the expression of SOX9 and type II collagen in chondrocytes; in areas away from the cartilage surface, the content of LAMP1 protein associated with burial in macrophages is higher than that in macrophages in areas close to the cartilage surface, TGF-β1 expression is lower than that in macrophages in areas close to the cartilage surface, and LRG1 expression is higher than that in macrophages in areas close to the cartilage surface.

[0091] Specifically, the apoptotic cell-pretreated macrophages are obtained by co-culturing bone marrow-derived macrophages and apoptotic cells at a ratio of 1:5.

[0092] Specifically, the macrophage efferocytosis achieves a dynamic balance of chondrogenic differentiation through the LRG1 / TGF-β1 bidirectional signaling axis: when the efferocytosis level is a ratio of bone marrow-derived macrophages to apoptotic cells of 1:5, the pro-chondrogenic axis TGF-β1 drives chondrogenic differentiation, and the expression of Sox9, Col2, and Acan increases, while inhibiting the expression of the fibrosis marker Col1.

[0093] Specifically, during joint development, macrophages in the central area away from the cartilage surface show high burial levels, driving high expression of LRG1, maintaining the morphology of the joint cavity by inhibiting cartilage differentiation; in the marginal area close to the cartilage surface, when the burial level is at a ratio of bone marrow-derived macrophages and apoptotic cells of 1:5, TGF-β1 expression is induced, promoting chondrogenic differentiation and matrix production of chondrogenic progenitor cells.

[0094] Based on the same inventive concept, an embodiment of the present invention further proposes a macrophage preparation pretreated with apoptotic cells, which is used to prepare a preparation for treating osteoarthritis. The macrophages pretreated with apoptotic cells are used to alleviate the inflammatory effects of osteoarthritis and promote cartilage regeneration in osteoarthritis, including promoting the expression of SOX9 and type II collagen in chondrocytes; in areas away from the cartilage surface, the content of LAMP1 protein associated with burial in macrophages is higher than that in macrophages in areas close to the cartilage surface, the expression of TGF-β1 is lower than that in macrophages in areas close to the cartilage surface, and the expression of LRG1 is higher than that in macrophages in areas close to the cartilage surface.

[0095] Specifically, the apoptotic cell-pretreated macrophages are obtained by co-culturing bone marrow-derived macrophages and apoptotic cells at a ratio of 1:5.

[0096] Based on the same inventive concept, an embodiment of the present invention further provides a method for preparing the above-mentioned macrophage preparation pretreated with apoptotic cells, the method comprising: S1, Obtain bone marrow-derived macrophages and apoptotic cells.

[0097] S2, co-culturing the bone marrow-derived macrophages and apoptotic cells according to a preset ratio to obtain a macrophage preparation pretreated with apoptotic cells.

[0098] Specifically, step S1 includes: Add fetal bovine serum (FBS) and penicillin-streptomycin solution (PS) to α-MEM medium to a volume concentration of 10% FBS and 1% PS, respectively. Add macrophage colony-stimulating factor (M-CSF) to a final concentration of 50 ng / mL to obtain macrophage differentiation medium. Extract primary bone marrow cells and culture for about 8-12 hours. After the mesenchymal stem cells adhere to the wall, remove the unattached cells and transfer them to a regular culture dish. Add 10 mL of macrophage differentiation medium and culture in a cell culture incubator at 37°C and 5% CO2. After 7 days of continuous culture, the mononuclear precursor cells differentiated into macrophages, and bone marrow-derived macrophages were obtained; Transfer the Jurkat cell suspension to a centrifuge tube, centrifuge at 1200 rpm for 5 minutes, and discard the supernatant; resuspend with PBS and inoculate in a common culture dish to a cell inoculation density of 1×105 cells / mL; irradiate with 254 nm ultraviolet light for 15 minutes; then incubate in PBS for 2-3 hours to obtain apoptotic cells.

[0099] Specifically, step S2 includes: Apoptotic cells were added to a BMDM culture dish in a differentiation culture at a ratio of bone marrow-derived macrophages: apoptotic cells = 1:5. After incubation at 37°C for 6 hours, the supernatant was discarded; after washing once with PBS, the dish was digested with 0.25% trypsin at 37°C for 2 minutes. The dish was gently tapped to observe that all cells floated and did not adhere to the wall. After that, 4 mL of proliferation medium was added to terminate the digestion and the liquid was collected in a centrifuge tube; the collected cell suspension was centrifuged at 1000 rpm for 5 minutes, and the supernatant was discarded; PBS was added to obtain a macrophage preparation pretreated with apoptotic cells.

[0100] The various embodiments in this specification are described in a progressive manner, and each embodiment focuses on the differences from other embodiments. The same or similar parts between the various embodiments can be referenced to each other.

[0101] Although the preferred embodiments of the present invention have been described, those skilled in the art may make additional changes and modifications to these embodiments once they have learned the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention.

[0102] Finally, it should be noted that, in the present invention, relational terms such as first and second, etc., are merely used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any actual relationship or order between these entities or operations. Moreover, the terms "include", "comprises", or any other variants thereof are intended to cover non-exclusive inclusion, so that a process, method, article, or terminal device that includes a series of elements includes not only those elements, but also other elements that are not explicitly listed, or also includes elements that are inherent to such process, method, article, or terminal device. In the absence of further restrictions, an element defined by the sentence "comprises a ..." does not exclude the presence of other identical elements in the process, method, article, or terminal device that includes the element.

[0103] The above describes in detail the use of macrophages pretreated with apoptotic cells in the preparation of osteoarthritis treatment preparations, cell preparations, and preparation methods provided by the present invention. Specific examples are used in the present invention to illustrate the principles and implementation methods of the present invention. The description of the above embodiments is only intended to help understand the methods and core concepts of the present invention. At the same time, for those skilled in the art, based on the concepts of the present invention, there may be changes in the specific implementation methods and application scopes. In summary, the contents of this specification should not be understood as limiting the present invention.

Claims

1. Use of macrophages pretreated with apoptotic cells in the preparation of a preparation for treating osteoarthritis, characterized in that: The macrophages pretreated with apoptotic cells are used to alleviate osteoarthritis inflammation and promote osteoarthritis cartilage regeneration, including promoting the expression of SOX9 and type II collagen in chondrocytes; in areas away from the cartilage surface, the content of LAMP1 protein associated with burial in macrophages is higher than that in macrophages in areas close to the cartilage surface, the expression of TGF-β1 is lower than that in macrophages in areas close to the cartilage surface, and the expression of LRG1 is higher than that in macrophages in areas close to the cartilage surface.

2. The use of macrophages pretreated with apoptotic cells according to claim 1 in preparing a preparation for treating osteoarthritis, characterized in that: The apoptotic cell-pretreated macrophages are obtained by co-culturing macrophages and apoptotic cells at a ratio of 1:1-10.

3. Use of the macrophages pretreated with apoptotic cells according to claim 2 in the preparation of a preparation for the treatment of osteoarthritis, wherein the macrophage efferocytosis achieves a dynamic balance of chondrogenic differentiation through a bidirectional signaling axis of LRG1 / TGF-β1: efferocytosis promotes the secretion of TGF-β1 by bone marrow-derived macrophages, driving increased expression of Sox9, Col2, and Acan and corresponding chondrogenic differentiation, while inhibiting the expression of the fibrosis marker Col1.

4. The use of macrophages pretreated with apoptotic cells according to claim 2 in preparing a preparation for treating osteoarthritis, characterized in that: During joint development, macrophages in the central area away from the cartilage surface show high levels of burial, driving high expression of LRG1, maintaining the morphology of the joint cavity by inhibiting cartilage differentiation; in the marginal area close to the cartilage surface, macrophages secrete TGF-β1 when burializing apoptotic cells, promoting chondrogenic differentiation and matrix production of chondrogenic progenitor cells.

5. A macrophage preparation pretreated with apoptotic cells, characterized in that: The macrophage preparation pretreated with apoptotic cells is used to prepare a preparation for treating osteoarthritis. The macrophages pretreated with apoptotic cells are used to alleviate osteoarthritis inflammation and promote osteoarthritis cartilage regeneration, including promoting the expression of SOX9 and type II collagen in chondrocytes. In areas away from the cartilage surface, the content of LAMP1 protein associated with burial in macrophages is higher than that in macrophages in areas close to the cartilage surface, the expression of TGF-β1 is lower than that in macrophages in areas close to the cartilage surface, and the expression of LRG1 is higher than that in macrophages in areas close to the cartilage surface.

6. The macrophage preparation pretreated with apoptotic cells according to claim 5, characterized in that The apoptotic cell-pretreated macrophages are obtained by co-culturing macrophages and apoptotic cells at a ratio of 1:1-10.

7. The method for preparing the macrophage preparation pretreated with apoptotic cells according to claim 2 or 3, characterized in that: The method comprises: Macrophages and apoptotic cells are co-cultured according to a preset ratio to obtain a macrophage preparation pretreated with apoptotic cells.

8. The method for preparing the macrophage preparation pretreated with apoptotic cells according to claim 7, characterized in that: The method further comprises: Fetal bovine serum (FBS) and penicillin-streptomycin solution (PS) were added to α-MEM medium to make the volume concentrations of FBS and PS 10% and 1%, respectively. Macrophage colony-stimulating factor (M-CSF) was added to make the final concentration 50 ng / mL to obtain a macrophage differentiation medium. Extract primary bone marrow cells and culture for about 8-12 hours. After the mesenchymal stem cells adhere to the wall, remove the unattached cells and transfer them to a regular culture dish. Add 10 mL of macrophage differentiation medium and culture in a cell culture incubator at 37°C and 5% CO2. After 7 days of continuous culture, the mononuclear precursor cells differentiated into macrophages, and bone marrow-derived macrophages were obtained; Transfer the Jurkat cell suspension to a centrifuge tube, centrifuge at 1200 rpm for 5 minutes, discard the supernatant, resuspend with PBS, and inoculate in a common culture dish to a cell inoculation density of 1×10 5 cells / mL; irradiated with 254 nm ultraviolet light for 15 minutes; and then incubated in PBS for 2-3 hours to obtain apoptotic cells.

9. The method for preparing the macrophage preparation pretreated with apoptotic cells according to claim 7, characterized in that: Macrophages are co-cultured with apoptotic cells according to a preset ratio to obtain a macrophage preparation pretreated with apoptotic cells, comprising: Apoptotic cells were added to the BMDM culture dish in differentiation culture at a preset ratio. After incubation at 37°C for 6 hours, the supernatant was discarded. After washing once with PBS, the cells were digested with 0.25% trypsin at 37°C for 2 minutes. The culture dish was gently tapped to observe that all the cells were floating and not attached to the wall. After that, 4 mL of proliferation medium was added to terminate the digestion and the liquid was collected in a centrifuge tube. The collected cell suspension was centrifuged at 1000 rpm for 5 minutes and the supernatant was discarded. PBS was added to obtain a macrophage preparation pretreated with apoptotic cells.

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