A method for constructing osteophyte organoids and application thereof

Abstract

This invention provides a method for constructing osteophyte organoids and their applications, belonging to the field of cell culture technology. The method includes: centrifuging cartilage stem cells to obtain cell aggregates; culturing the cell aggregates statically, followed by osteogenic induction culture to obtain osteophyte organoids. This invention, through centrifugation and static treatment, induces cartilage stem cells to form aggregate structures, and further combines this with osteogenic induction culture, allowing the cartilage stem cell aggregates to undergo an endochondral ossification-like process, ultimately forming organoids with typical pathological features of osteophytes. In vitro experiments have verified that these osteophyte organoids can effectively evaluate the preventive and therapeutic effects of anti-osteophyte drugs, and can serve as an effective experimental platform for studying the pathological mechanisms of osteophytes related to osteoarthritis and screening anti-osteophyte drugs, possessing significant scientific research value and social significance.

Description

Technical Field

[0001] This invention belongs to the field of cell culture technology, specifically relating to a method for constructing osteophyte organoids and its application. Background Technology

[0002] Osteophytes, commonly known as bone spurs, are a typical pathological change in degenerative joint diseases such as osteoarthritis. Essentially, osteophytes are ectopic bone formation at the joint margins. Their pathological process involves abnormal hypertrophy of chondrocytes, calcification of the cartilage matrix, and subsequent osteogenic differentiation, specifically the ectopic activation of endochondral ossification (EO). Osteophyte formation is closely related to various factors such as articular cartilage degeneration, abnormal biomechanical stress, and synovial inflammation. Patients often experience clinical symptoms such as local masses, pain, and limited mobility. Clinically, there is still a lack of effective specific treatments for osteophytes, mainly due to insufficient understanding of the molecular pathological mechanisms of osteophyte formation and the lack of reliable in vitro research models.

[0003] Currently, research on osteophytes mainly relies on two-dimensional cell culture and animal models. Two-dimensional monolayer culture cannot simulate the three-dimensional spatial structural relationships between cells and between cells and the extracellular matrix in vivo, making it difficult to truly reflect the complex microenvironment signal interactions during osteophyte formation. Although animal models can partially reproduce in vivo pathological processes, their high cost and low throughput make them unsuitable for large-scale drug screening and mechanism studies. Summary of the Invention

[0004] In view of this, the present invention provides a method for constructing osteophyte organoids. The prepared osteophyte organoids have typical pathological characteristics of osteophytes and can effectively evaluate the preventive and therapeutic effects of anti-osteophyte drugs.

[0005] To achieve the above objectives, the present invention provides the following technical solution: This invention provides a method for constructing osteophyte organoids, comprising the following steps: centrifuging cartilage stem cells to obtain cell aggregates; After static culture of the cell aggregates, osteogenic induction culture was performed to obtain osteophyte organoids.

[0006] Preferably, the density of the centrifuged chondrocyte stem cells is 1×10⁻⁶. 5 ~3×10 5 cells / mL.

[0007] Preferably, the centrifugation speed is 1000~1500 rpm and the centrifugation time is 3~7 min.

[0008] Preferably, the static incubation time is 24-60 h.

[0009] Preferably, the culture medium used for osteogenic induction includes an induction medium; the induction medium is based on DMEM medium and further includes the following components: dexamethasone 0.08~0.12 μM, sodium β-glycerophosphate 8~12 mM and bone morphogenetic protein 2 0.4~0.6 μg / mL.

[0010] Preferably, the culture medium used for osteogenic induction culture further includes fetal bovine serum; the volume ratio of the osteogenic induction culture medium to the fetal bovine serum is 8~10:1.

[0011] Preferably, the osteogenic induction culture time is 15-25 days; the osteogenic induction culture temperature is 36-38℃.

[0012] This invention provides osteophyte organoids prepared by the aforementioned construction method.

[0013] This invention provides the application of the osteophyte organoids in the preparation of pathological models of osteophyte lesions.

[0014] This invention provides the application of the aforementioned osteophyte organoids in screening and / or evaluating drugs; The medications include those for the prevention and / or treatment of osteophytes.

[0015] Compared with the prior art, the present invention has the following advantages: This invention provides a method for constructing osteophyte organoids, comprising the following steps: centrifuging cartilage stem cells to obtain cell aggregates; culturing the cell aggregates statically, followed by osteogenic induction culture to obtain osteophyte organoids. This invention, through centrifugation and static treatment, induces cartilage stem cells to form aggregate structures, and further combines this with osteogenic induction culture, allowing the cartilage stem cell aggregates to undergo an intrachondral ossification-like process, ultimately forming osteophyte organoids with typical pathological features. The osteophyte organoids prepared by the method of this invention exhibit irregularly invasive protrusions at the edges, high expression of Runt-related transcription factor 2 (RUNX2) and type I collagen (COL1), filling the gap in osteophyte-specific in vitro models. In vitro experiments have verified that these osteophyte organoids can effectively evaluate the preventive and therapeutic effects of anti-osteophyte drugs, and can serve as an effective experimental platform for studying the pathological mechanisms of osteophytes related to osteoarthritis and screening anti-osteophyte drugs, possessing significant scientific research value and social significance. Attached Figure Description

[0016] Figure 1 This is a timeline diagram of osteophyte organoid construction and intervention; 48 hours is designated as day 0, days 0-21 represent osteogenic induction, and days 22-35 represent maintenance culture or drug intervention. Figure 2Immunofluorescence and immunohistochemical staining images of RUNX2 and COL1 in osteophyte organoids are shown. A represents the fluorescence expression and quantitative data analysis results of RUNX2 (green) and DAPI (blue); B represents the expression distribution and quantitative data analysis results of COL1, with brown indicating positive staining. The scale bar in B is 100 μm. C represents the relative expression levels of RUNX2 and COL1 mRNA. Compared with the NC group, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. Figure 3 Immunofluorescence and immunohistochemical staining images of RUNX2 and COL1 in osteophyte organoids are shown. A represents the fluorescence expression and quantitative data analysis results of RUNX2 (red) and DAPI (blue); B represents the expression distribution and quantitative data analysis results of COL1, with brown indicating positive staining. Compared with the NC group, *P<0.05, **P<0.01; compared with the Model group, # P<0.05, ## P<0.01; the scale bars for A and B are 100 μm. Detailed Implementation

[0017] This invention provides a method for constructing osteophyte organoids, comprising the following steps: centrifuging cartilage stem cells to obtain cell aggregates; After static culture of the cell aggregates, osteogenic induction culture was performed to obtain osteophyte organoids.

[0018] This invention induces chondrocyte stem cells to form aggregate structures through centrifugation and static culture. Further osteogenic induction culture allows these aggregates to undergo an intrachondral ossification-like process, increasing COL1 expression levels, promoting osteogenic differentiation, and facilitating bone matrix maturation and mineralization, ultimately resulting in osteophyte-like structures with typical pathological features. The osteophyte organoids prepared using the method described in this invention exhibit irregular, invasive protrusions at the edges, high expression of RUNX2 and COL1, and, under the same induction conditions, significantly increased COL1 expression compared to two-dimensional cells, filling a gap in osteophyte-specific in vitro models. In vitro experiments have verified that these osteophyte organoids can effectively evaluate the preventive and therapeutic effects of anti-osteophyte drugs, serving as an effective experimental platform for studying the pathological mechanisms of osteoarthritis-related osteophytes and screening anti-osteophyte drugs, demonstrating significant scientific research value and social significance.

[0019] In this invention, the chondrocyte stem cells are preferably derived from articular cartilage tissue, more preferably from rat articular cartilage tissue. The chondrocyte stem cells preferably include chondrocyte stem cells passaged 2-5 times, more preferably including chondrocyte stem cells passaged 3 times. These chondrocyte stem cells are more conducive to forming stable cell aggregates during subsequent centrifugation, and more conducive to the intrachondral ossification process during subsequent osteogenic induction culture, ultimately forming osteophyte-like structures. In this invention, the density of the centrifuged chondrocyte stem cells is preferably 1×10⁻⁶. 5 ~3×10 5 cells / mL, more preferably 1.5 × 10⁻⁶ cells / mL. 5 ~2.9×10 5 cells / mL, more preferably 2.86 × 10⁻⁶. 5 The number of chondrocytes processed by centrifugation is preferably 2 × 10⁻⁶ cells / mL. 5 The preferred centrifugation volume is 700 μL. The preferred centrifugation speed is 1000-1500 rpm, which can be 1200, 1300, or 1400 rpm. The preferred centrifugation time is 3-7 min, more preferably 4-6 min, and even more preferably 5 min. Excessive or insufficient centrifugation force will not yield osteophyte organoids with high COL1 expression; insufficient centrifugation force will not form spherical structures, while excessive centrifugation force will reduce cell viability and affect bone differentiation. The cell density, cell number, and centrifugation parameters are more conducive to promoting the formation of cell aggregates of moderate density and size in chondrocyte stem cells, which is more conducive to the subsequent endochondral ossification process during osteogenic induction culture, increasing COL1 expression levels, promoting osteogenic differentiation, promoting bone matrix maturation and mineralization, entering the functional osteogenic stage, and ultimately forming osteophyte-like structures with typical pathological features. This invention demonstrates that centrifugation causes cells to aggregate into three-dimensional cell spheres, which is more conducive to osteogenic differentiation, and the COL1 content in three-dimensional cell spheres is significantly increased compared to planar structures.

[0020] In this invention, the static culture time is preferably 24-60 h, more preferably 36-55 h, and even more preferably 48 h. Static culture is more conducive to the formation of stable three-dimensional spherical structures of cell aggregates and facilitates the subsequent endochondral ossification process during osteogenic induction culture, resulting in better bone differentiation of internal cells, a significant increase in COL1, and ultimately a result closer to the typical pathological characteristics of osteophytes. Without static culture, a three-dimensional spherical structure cannot be obtained, and under the same induction conditions, COL1 is significantly reduced compared to a three-dimensional spherical structure.

[0021] In this invention, the culture medium used for osteogenic induction culture preferably includes an induction medium; the culture medium also preferably includes fetal bovine serum. The volume ratio of the osteogenic induction medium to the fetal bovine serum is preferably 8-10:1, more preferably 9:1. The induction medium is based on DMEM medium and preferably includes the following components: dexamethasone 0.08-0.12 μM, sodium β-glycerophosphate 8-12 mM, and bone morphogenetic protein 2 (BMP-2) 0.4-0.6 μg / mL. The concentration of dexamethasone is more preferably 0.1 μM; the concentration of sodium β-glycerophosphate is more preferably 10 mM; and the concentration of BMP-2 is more preferably 0.5 μg / mL. The culture medium used for osteogenic induction culture is more conducive to promoting the intrachondral ossification process of cell aggregates, ultimately forming osteophytes with typical pathological features. The osteogenic induction culture time is preferably 15-25 days, more preferably 18-23 days, and more preferably 21 days. The preferred temperature for osteogenic induction culture is 36-38℃, more preferably 37℃. This induction method is more conducive to promoting the endochondral ossification process of cell aggregates, ultimately forming osteophytes with typical pathological features.

[0022] This invention provides osteophyte organoids prepared by the aforementioned construction method.

[0023] The osteophyte organoids described in this invention have typical pathological features of osteophytes. As demonstrated by the embodiments of this invention, the osteophyte organoids have a three-dimensional spherical structure with irregular protrusions at the edges; immunofluorescence staining shows that RUNX2 is highly expressed and located in the cell nucleus, and COL1 is positively highly expressed.

[0024] Based on the fact that the osteophyte organoids possess typical pathological features of osteophytes, this invention provides the application of the osteophyte organoids in preparing pathological models of osteophyte lesions. The osteophyte lesions may include osteoarthritis-related osteophyte lesions.

[0025] This invention provides the use of the osteophyte organoids in screening and / or evaluating drugs; the drugs include those for the prevention and / or treatment of osteophytes.

[0026] In this invention, the method for screening and / or evaluating drugs for preventing and / or treating osteophytes preferably includes: culturing the osteophyte organoids in a drug-containing culture medium, and comparing them with osteophyte organoids cultured in a drug-free culture medium, assessing the drug's effectiveness in preventing and / or treating osteophytes based on the expression levels of RUNX2 and COL1. If, after administration, the expression levels of RUNX2 and COL1 in the osteophyte organoids decrease, it indicates that the drug has a therapeutic effect on osteophytes. The drug preferably includes traditional Chinese medicine compound formulas, chemically synthesized drugs, and biological agents. The traditional Chinese medicine compound formula is preferably in the form of drug-containing serum. Drug-containing serum can more accurately reflect the effective substances that actually exert the drug's effects in vivo. The drug-containing serum is preferably prepared by collecting blood and separating serum from rats after oral administration of the drug.

[0027] In this invention, Xibi Decoction and celecoxib are drugs used to treat osteophytes. The embodiments of this invention use the described method to evaluate the efficacy of the drugs, and the results show that both Xibi Decoction and celecoxib have therapeutic effects. Therefore, the method of this invention can accurately evaluate and screen drugs for treating osteophytes.

[0028] To further illustrate the present invention, the solutions provided by the present invention will be described in detail below with reference to the accompanying drawings and embodiments, but these should not be construed as limiting the scope of protection of the present invention.

[0029] 1. Reagent preparation Dexamethasone stock solution (1 mM): Accurately weigh 0.392 mg of dexamethasone powder, dissolve it in 1 mL of anhydrous DMSO, aliquot and store at -20 ℃.

[0030] β-Glycerophosphate stock solution (1 M): 1 M β-glycerophosphate sodium solution was prepared with deionized water, sterilized by filtration through a 0.22 μm filter membrane, aliquoted, and stored at -20 ℃.

[0031] BMP-2 stock solution (20 μg / mL): Dissolve 10 μg of BMP-2 lyophilized powder in 500 μL of sterile PBS buffer containing 0.1% (w / v) bovine serum albumin, aliquot, and store at -80 °C.

[0032] The preparation method of serum containing Xibi Decoction is disclosed in Example 3 of Patent CN 121513082 A, which describes the preparation of serum containing Xibi Decoction.

[0033] Fuzi decoction-containing serum: The preparation method is disclosed in the existing technology (Chen Z, Zhou L, Ge Y, Chen J, Du W, Xiao L, Tong P, Huang J, Shan L, Efferth T. Fuzi decoction ameliorates pain and cartilage degeneration of osteoarthritic rats through PI3K-Akt signaling pathway and its clinical retrospective evidence. Phytomedicine. 2022 Jun;100:154071. doi: 10.1016 / j.phymed.2022.154071. Epub 2022 Mar 20. PMID: 35378415.) under Preparation of FZD-containing serum (FCS); Celecoxib working solution: Accurately weigh 381.4 mg of celecoxib powder and dissolve it in 10 mL of DMSO to prepare a 100 mM stock solution. Take 2 μL of the stock solution and dilute it to 2 mL with serum-free DMEM medium to obtain a 100 μM intermediate working solution.

[0034] Normal group culture medium: 10% (v / v) fetal bovine serum (FBS) was added to DMEM medium; DMEM medium was purchased from HyClone.

[0035] Model group induction medium: 10% FBS was added to DMEM medium containing 0.1 μM dexamethasone, 10 mM sodium β-glycerophosphate and 0.5 μg / mL BMP-2.

[0036] DMEM medium containing 10% Xibi Decoction-containing serum: Add 10% (v / v) of Xibi Decoction-containing serum to DMEM medium; Induction medium for the model group containing 10% Xibi Decoction-containing serum: 10% Xibi Decoction-containing serum was added to DMEM medium containing 0.1 μM dexamethasone, 10 mM β-glycerophosphate sodium and 0.5 μg / mL BMP-2. DMEM medium containing 10% Fuzi decoction-containing serum: Add 10% (v / v) of Fuzi decoction-containing serum to DMEM medium; Celecoxib treatment group culture medium (20 μM): Take 2 mL of the intermediate working solution, 7.2 mL of DMEM culture medium and 0.8 mL of FBS, and mix well.

[0037] Example 1: Preparation and Identification of Osteophyte Organoids 1. Isolation and culture of chondrocyte stem cells Knee joint tissue was taken from 8-week-old SD rats and cut into pieces of approximately 1 mm using ophthalmic scissors. 3 Small pieces of cells were added to 0.1% (w / v) type II collagenase (purchased from Roche) and digested overnight in a metal bath at 37 ℃. After digestion, the cells were filtered through a 100-mesh cell sieve, and the filtrate was centrifuged at 1000 rpm for 5 min. The supernatant was discarded, and the cells were resuspended in DMEM medium containing 10% (v / v) FBS and 1% (v / v) penicillin-streptomycin. The cell suspension was seeded into T25 culture flasks and cultured in a 37 ℃, 5% CO2 incubator. When the cell confluence reached 80%–90%, the cells were digested and passaged using 0.25% (w / v) trypsin-EDTA digestion solution, and the third-generation chondrocyte stem cells were used for subsequent experiments.

[0038] 2. Preparation method of three-dimensional cell spheres Third-generation chondrocyte stem cells were digested and counted, and the cell density was adjusted to 1×10⁻⁶ using normal group culture medium. 6 Cells were counted at a density of 10 cells / mL to obtain a cell suspension. Take 1.5 mL EP tubes, add 500 μL of normal group culture medium to each tube, then add 200 μL of cell suspension (i.e., 2 × 10⁻⁶ cells / mL). 5 (Cells). Place the EP tube in a centrifuge and centrifuge at 1000 rpm for 5 min to allow the cells to aggregate into three-dimensional cell spheres. After centrifugation, carefully transfer the EP tube along with the cells to a 37 ℃, 5% CO2 incubator for culture. After 48 h, the cell clusters will spontaneously reassemble into cell spheres. Transfer the three-dimensional cell spheres to 96-well low-adsorption culture plates for induction culture.

[0039] 3. Induction Culture The three-dimensional cell spheres were divided into a model group and a normal group.

[0040] Model group: Three-dimensional cell spheres were induced and cultured using the model group induction medium throughout the entire process (days 1-21). The medium was changed every 3 days to obtain induced cell spheres. The culture conditions were 37 ℃ and 5% CO2 incubator.

[0041] Normal group (NC): The only difference from the model group is that the model group's induction medium is replaced with the normal group's medium.

[0042] 4. Identification of osteophyte organoids (1) Immunofluorescence staining: After induction culture, cell spheres from each group were collected and fixed overnight with 4% (w / v) paraformaldehyde. After dehydration with 30% (w / v) sucrose until the cell spheres sank to the bottom of the EP tube, they were embedded with OCT embedding agent, frozen, and sectioned to a thickness of 10 μm. The sections were permeabilized with immunostaining permeabilization buffer (Beyotime) for 5 min, blocked with blocking buffer (Zhongshan Jinqiao) at room temperature for 1 h, and rabbit anti-RUNX2 antibody (1:200 dilution, Abcam) was added and incubated overnight at 4 ℃ in the dark. The next day, goat anti-rabbit IgG labeled with Alexa Fluor 488 or Alexa Fluor 570 (purchased from Zhongshan Jinqiao) was added and incubated at room temperature in the dark for 1 h. DAPI was added and incubated at room temperature in the dark for 4 min to stain the nuclei. The sections were mounted with anti-fluorescence quenching mounting medium (Beyotime). The images were observed and acquired under a fluorescence microscope. The results are shown in the figure. Figure 2 Middle A (goat anti-rabbit IgG labeled with Alexa Fluor 488).

[0043] The results showed that RUNX2 was strongly expressed in the cell spheres of the model group and located in the cell nucleus; RUNX2 expression was very weak in the normal control group. RUNX2 positivity indicates the initiation of osteogenic / osteophyte formation. This result indicates that the osteophyte organoid model was successfully constructed and the model group was successfully induced to exhibit an osteophyte-like pathological phenotype.

[0044] (2) Immunohistochemical staining: The procedures for paraformaldehyde fixation, embedding, and sectioning were the same as for immunofluorescence staining. Sections were permeabilized with immunostaining permeabilization buffer (Beyotime) for 10 min, then infiltrated with endogenous peroxidase inhibitor (Zhongshan Jinqiao) for 10 min, and blocked with blocking buffer (Zhongshan Jinqiao) at room temperature for 1 h. Rabbit anti-COL1 antibody (1:200 dilution, Abcam) was added, and incubated overnight at 4℃. The next day, the primary antibody was recovered, and reaction enhancement buffer (Zhongshan Jinqiao) was added. After incubation at 37℃ for 20 min, enzyme-labeled goat anti-rabbit IgG polymer (Zhongshan Jinqiao) was added, and incubation continued at 37℃ for 20 min. DAB chromogenic reagent kit (Zhongshan Jinqiao) was used for chromogenic treatment. After chromogenic treatment, counterstaining was performed sequentially: hematoxylin staining for 40 s, followed by washing with water; hydrochloric acid alcohol differentiation for 3 s, followed by washing with water; 0.5% ammonia solution for blueing for 10 s, followed by washing with water. Finally, the sections were mounted with neutral resin, observed under an optical microscope, and images were acquired. The results are shown in […]. Figure 2 B.

[0045] The results showed that COL1 was positively expressed in the cell spheroids of the model group, while COL1 expression was very weak in the normal group. COL1 positivity indicates that the bone matrix has matured and mineralized, and has entered the functional osteogenic stage. This result shows that the osteophyte organoid model has successfully completed the entire process of endochondral ossification, not only initiating at the molecular regulatory level (RUNX2), but also reconstructing the bone matrix characteristics of the osteophyte at the tissue structure level (COL1).

[0046] (3) Real-time quantitative PCR (qPCR): Cell spheres induced and cultured in each group were collected, and total RNA was extracted with TRIzol and reverse transcribed into cDNA. Actin was used as an internal control to detect the mRNA expression levels of RUNX2 and COL1. Primer sequences are shown in Table 1.

[0047] Table 1 Primer sequences of the target gene

[0048] Table 2. mRNA expression levels of various osteogenic marker genes

[0049] The results are shown in Table 2 and Figure 2 The results showed that the mRNA expression levels of each osteogenic marker gene in the model group were significantly higher than those in the normal group. This result indicates that an organoid model with osteophyte pathological characteristics was successfully prepared.

[0050] Comparative Example 1 The preparation method of the three-dimensional cell spheres was the same as that in Example 1. 500 μL of normal group culture medium was not added during centrifugation. The centrifugation was carried out in a 96-well low-adsorption culture plate with a centrifugal force of 200 g.

[0051] After induction culture, cell structure was observed. The expression of RUNX2 in each group was detected by immunofluorescence staining method in Example 1, and the expression of COL1 was detected by immunohistochemical staining method in Example 1. The results showed that with low centrifugal force, cells did not form spherical structures. Compared with the normal group in Example 1, the expression levels of RUNX2 and COL1 were significantly increased, and the expression level of COL1 was significantly lower than that of the model group in Example 1.

[0052] Comparative Example 2 The third-generation chondrocytes obtained using the method in Example 1 were directly used for induction culture without the preparation of three-dimensional cell spheres.

[0053] After induction culture, cell structure was observed. RUNX2 in each group was detected by immunofluorescence staining method in Example 1, and COL1 expression was detected by immunohistochemical staining method in Example 1. The results showed that the cells did not form a spherical structure. Compared with the normal group in Example 1, the expression levels of RUNX2 and COL1 were significantly increased, and the expression level of COL1 was significantly lower than that of the model group in Example 1.

[0054] Example 2: Application of osteophyte organoids in the screening of anti-osteophyte drugs (prevention and treatment evaluation) 1. Experimental grouping and treatment After preparing the induced cell spheres according to the method in Example 1, the following 6 groups (3 cell spheres in each group) were set up: Normal group (NC): Normal group culture medium was used throughout the entire course (days 1-35).

[0055] Model group: osteophyte model was constructed using model group induction medium from day 1 to day 21, and normal group medium was used from day 22 to day 35.

[0056] The Xibi Decoction treatment group (BSMG-ZL): osteophyte models were constructed using the model group induction medium from day 1 to day 21, and DMEM medium containing 10% Xibi Decoction-containing serum was used from day 22 to day 35.

[0057] Xibi Decoction Prevention Group (BSMG-YF): The model group was induced with serum containing 10% Xibi Decoction throughout the entire course (days 1-21).

[0058] Fuzi decoction treatment group (FZD): osteophyte model was constructed using the model group induction medium from day 1 to day 21, and DMEM medium containing 10% Fuzi decoction-containing serum was used from day 22 to day 35.

[0059] Celecoxib treatment group (CELE): osteophyte model was constructed using the model group induction medium from day 1 to day 21, and the celecoxib treatment group medium was used from day 22 to day 35.

[0060] All culture media were replaced every 3 days at a rate of 200 μL per well, and the culture conditions were 37 °C and 5% CO2. Figure 1 A timeline diagram illustrating the construction and intervention of osteophyte organoids.

[0061] 2. Experimental Results After 35 days of culture, the expression of RUNX2 in each group was detected by immunofluorescence staining method in Example 1, and the expression of COL1 was detected by immunohistochemical staining method in Example 1, in order to evaluate the anti-osteophyte effect of the drug.

[0062] The results of immunofluorescence staining for RUNX2 are shown in the figure. Figure 3 The results showed that the RUNX2 fluorescence intensity was strongest in the model group; the fluorescence intensity of each prevention and treatment group was weaker than that of the model group, with the Xibi Decoction treatment group and the celecoxib group showing the most significant effects, while the Xibi Decoction prevention group had comparable effects to its treatment group.

[0063] The results of immunohistochemical staining for COL1 are shown in the figure. Figure 3The results showed that the model group had the highest percentage of COL1 positive area, while the normal group had the lowest. The Xibi Decoction prevention group and the celecoxib group were comparable to the model group, indicating that osteophyte organoids can be used to evaluate the anti-osteophyte effect of drugs. Furthermore, the Xibi Decoction treatment group had a similar effect to the NC group, suggesting that Xibi Decoction may be more suitable for the treatment of mid-to-late stage osteoarthritis than for early prevention.

[0064] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. Other embodiments can be obtained based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A method for constructing osteophyte organoids, characterized in that, Includes the following steps: Cartilage stem cells were centrifuged to obtain cell aggregates; After static culture of the cell aggregates, osteogenic induction culture was performed to obtain osteophyte organoids.

2. The construction method according to claim 1, characterized in that, The density of the centrifuged chondrocyte stem cells is 1 x 10 5 3 x 10 5 cells / mL.

3. The construction method according to claim 1, characterized in that, The centrifugation speed is 1000~1500 rpm, and the centrifugation time is 3~7 min.

4. The construction method according to claim 1, characterized in that, The static incubation time is 24-60 hours.

5. The construction method according to claim 1, characterized in that, The culture medium used for osteogenic induction includes an induction medium; the induction medium is based on DMEM medium and also includes the following components: dexamethasone 0.08~0.12 μM, sodium β-glycerophosphate 8~12 mM and bone morphogenetic protein 2 0.4~0.6 μg / mL.

6. The construction method according to claim 5, characterized in that, The culture medium used for osteogenic induction culture also includes fetal bovine serum; the volume ratio of the osteogenic induction culture medium to the fetal bovine serum is 8~10:

1.

7. The construction method according to any one of claims 1 to 6, characterized in that, The osteogenic induction culture time is 15-25 days; the osteogenic induction culture temperature is 36-38 ℃.

8. Osteophyte organoids prepared by the construction method according to any one of claims 1 to 7.

9. The application of the osteophyte organoids of claim 8 in the preparation of pathological models of osteophyte lesions.

10. The use of the osteophyte organoids of claim 8 in screening and / or evaluating drugs; The medications include those for the prevention and / or treatment of osteophytes.

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