Construction method and application of callus-like organ based on intra-cartilage ossification

Abstract

The invention discloses a cartilage internal ossification-based callus-like organ construction method and application. The construction method comprises the following steps: putting hydrogel microspheres loaded with stem cells into cartilage induction liquid for culturing to obtain cartilage spheres; and then putting the cartilage balls into the cartilage induction liquid containing bioactive glass for continuous culture to obtain the cartilage balls. The cartilage internal ossification-based callus-like organ construction method is simple and low in cost, the prepared callus-like organ has good osteogenic differentiation and matrix mineralization capabilities, and the healing time of large bone defects can be effectively shortened when the callus-like organ is applied to bone defect treatment.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a method and application of constructing callus organoids based on endochondral ossification. Background Art

[0002] Bone defect refers to the phenomenon that the structural integrity of the bone is destroyed, resulting in partial bone loss. A small part of the bone defect can be repaired by itself, but if the defect is too long or too large, it will lead to non-healing of the defect site, which is a common clinical disease. At present, the main methods for repairing large-area bone defects in clinical practice are metal grafts, autologous bone grafts, allogeneic bone grafts and artificial bone grafts. Among them, metal grafts can be customized according to the patient's bone defect site; autologous bone grafts can avoid the risk of immune rejection because they use autologous bone; allogeneic bone grafts help overcome the problem of insufficient autologous bone donors and do not need to traumatize other parts of the patient; the bone materials used in artificial bone grafts can be designed according to needs, which helps promote bone healing. However, these treatment methods have their disadvantages, such as the removal of metal devices may cause secondary damage, the application of autologous grafts opens up a new surgical area, and allogeneic grafts may increase the risk of immune rejection and disease transmission. In addition, these transplantation methods are difficult to significantly reduce the healing time of large bone defects. Therefore, it is urgent to seek more effective strategies to solve the above problems.

[0003] In the related technology, bone organoids are three-dimensional self-renewing, self-organizing micro-bone tissues with biomimetic spatial characteristics that are formed by directed differentiation of stem cells and constructed based on bioactive materials. Among them, the construction of callus organoids is inspired by the physiological process of endochondral ossification in the repair of large bone defects, and is more tolerant to the ischemic and hypoxic environment of large bone defects than other bone organoids. The construction of callus organoids is a continuous multi-stage induction process, including the use of mesenchymal stem cells to actively condense and differentiate at the defect site to form a cartilage template, followed by hypertrophy, calcification, and final osteogenic differentiation. At present, the construction of callus organoids mainly relies on a 3D culture environment, such as the use of hydrogel microspheres loaded with bone marrow mesenchymal stem cells (BMSC) to achieve callus organoid construction. Although it can effectively reduce the healing time of large bone defects compared to conventional bone transplantation methods, the actual osteogenic differentiation and matrix mineralization capabilities are poor, and it is difficult to meet actual needs.

[0004] Based on this, there is still a need to seek a method for constructing callus organoids that can not only effectively reduce the healing time of large bone defects but also have good osteogenic differentiation and matrix mineralization capabilities. Summary of the invention

[0005] The present invention aims to solve at least one of the technical problems existing in the prior art. To this end, the present invention proposes a method for constructing callus organoids based on endochondral ossification, which can be constructed in a simple and low-cost manner, and the prepared callus organoids have good osteogenic differentiation and matrix mineralization capabilities. Applying it to the treatment of bone defects can effectively reduce the healing time of large bone defects.

[0006] The present invention also provides a callus organoid.

[0007] The present invention also proposes a method for constructing the above-mentioned callus organoids based on endochondral ossification and the use of callus organoids in the preparation of bone defect repair products or drug screening.

[0008] A first aspect of the present invention provides a method for constructing callus organoids based on endochondral ossification, comprising the following steps: The hydrogel microspheres loaded with stem cells are placed in a cartilage induction solution for culture to obtain cartilage spheres; and then the cartilage spheres are placed in the cartilage induction solution containing bioactive glass for further culture to obtain the product.

[0009] The method for constructing callus organoids according to the embodiment of the present invention has at least the following beneficial effects: The present invention utilizes stem cells to sequentially induce the generation of self-mineralized callus organoids in vitro. The construction method is simple and low-cost, and the prepared callus organoids have good osteogenic differentiation and matrix mineralization capabilities. Applying them to the treatment of bone defects can effectively reduce the healing time of large bone defects.

[0010] In addition, the construction process of the callus organoids of the present invention utilizes three-dimensional culture technology to allow stem cells to grow in an environment similar to natural bone, which can better mimic the complex structure and physiological function of bone tissue, and bioactive glass is added during the construction process. It has good biological activity and helps to promote the hypertrophy of chondrocytes. In human body fluids, it can quickly induce the production of hydroxyapatite layers and release various ions to stimulate tissue cells, induce bone tissue growth and rapid mineralization, and help promote bone regeneration.

[0011] In some embodiments of the present invention, the method for preparing the hydrogel microspheres loaded with stem cells comprises: mixing and culturing the stem cells and the hydrogel microspheres in a complete culture medium until the surface growth density reaches 60% to 80%.

[0012] When the surface growth density is 60%~80% (preferably 65%~75%), it helps to promote cell-to-cell interactions, allowing cells to contact and communicate through secreted signaling molecules, which is crucial for cell differentiation and organoid formation. If the cell density is too low, the interaction between cells will be reduced, affecting their normal growth and differentiation process, and it will be difficult to effectively self-organize into complex three-dimensional structures; when the cell density is too high, excessive competition will occur between cells, leading to insufficient nutrients, oxygen and growth factors, affecting cell proliferation and differentiation, and then leading to incomplete development of organoids or even cell death.

[0013] In some embodiments of the present invention, the complete culture medium comprises 8% to 12% FBS, 0.5% to 1.5% double antibody and DMEM high glucose medium.

[0014] In some embodiments of the present invention, the stem cells include totipotent stem cells or pluripotent stem cells.

[0015] In some embodiments of the present invention, the pluripotent stem cells include bone marrow mesenchymal stem cells and / or bone marrow stem cells.

[0016] In some embodiments of the present invention, the conditions for mixed culture of the stem cells and the hydrogel microspheres are 37±2° C. and 4%~6% CO 2 .

[0017] In some embodiments of the present invention, the stem cells and hydrogel microspheres are mixed and cultured for 5 to 14 days.

[0018] In some embodiments of the present invention, the stem cells and the hydrogel microspheres are co-cultured and half of the medium is replaced every 1 to 2 days.

[0019] In some embodiments of the present invention, the hydrogel microspheres include at least one of methacrylated gelatin (GelMA) hydrogel microspheres, hyaluronic acid hydrogel microspheres, chitosan hydrogel microspheres, sodium alginate hydrogel microspheres, and gelatin hydrogel microspheres.

[0020] In some embodiments of the present invention, the diameter of the hydrogel microspheres is 100-1000 μm.

[0021] In some preferred embodiments of the present invention, the diameter of the hydrogel microspheres is 300-600 μm.

[0022] In some embodiments of the present invention, the hydrogel microspheres include methacrylated gelatin hydrogel microspheres.

[0023] Compared with other hydrogel microspheres, methacryloyl gelatin (GelMA) hydrogel microspheres have excellent biocompatibility. They utilize the cross-linking reaction of methacrylamide to enhance their stability and retain the natural properties of gelatin. In addition, a significant advantage of methacryloyl gelatin hydrogel microspheres is that they can promote the attachment, proliferation and differentiation of osteoblasts. As a natural protein source, gelatin has a peptide sequence similar to bone matrix (such as RGD sequence), which can effectively interact with osteoblasts (such as osteoblast precursor cells, osteoblasts), promote the functionalization of bone cells, and prevent the degeneration of chondrocytes on the plane. In addition, GelMA hydrogel microspheres can not only degrade well in the body, but also the degradation process usually does not produce toxic byproducts, which is very suitable for bone repair materials.

[0024] In some embodiments of the present invention, the method for preparing the methacrylylated gelatin hydrogel microspheres comprises: Disperse methacrylate gelatin in water, heat to dissolve, add phenyl (2,4,6-trimethylbenzoyl) phosphate lithium salt to obtain solution A; dissolve sorbitan oleate in isopropyl ester to prepare solution B; then drop solution A into solution B at a rate of 150-250 μL / min to obtain the solution.

[0025] In some embodiments of the present invention, the concentration of methacrylate gelatin in the solution A is 15% to 25%, and the concentration of phenyl (2,4,6-trimethylbenzoyl) phosphate lithium salt is 0.2% to 0.3%.

[0026] In some embodiments of the present invention, in the B solution, the mass volume ratio of the sorbitan oleate to the isopropyl ester is 1-5 g:100 mL.

[0027] In some embodiments of the present invention, the chondrology induction solution comprises 8%~12% fetal bovine serum, 0.5%~1.5% penicillin-streptomycin double antibody, 80~120 nM dexamethasone, 0.1~0.3 mM L-ascorbic acid-2-phosphate trisodium salt, 30~50 μg / mL proline, 0.8~1.2 mM sodium pyruvate, 0.8%~1.2% ITS, 8~12 ng / ml TGFβ3 and DMEM high glucose medium.

[0028] In some embodiments of the present invention, the culture conditions are 37±2°C and 4%~6% CO2.

[0029] In some embodiments of the present invention, the culture time is 12 to 18 days. Preferably, the culture time is 13 to 15 days.

[0030] In some embodiments of the present invention, half of the medium is replaced every 1 to 2 days during the culture process.

[0031] In some embodiments of the present invention, the method for preparing the bioactive glass comprises: Under solvent conditions, dodecylamine, ethyl silicate, triethyl phosphate and calcium nitrate tetrahydrate are added and mixed to react to obtain a precursor, which is then centrifuged and washed and calcined at 600-700°C for 2.5-3.5 h to obtain the product.

[0032] In some embodiments of the present invention, the solvent is selected from at least one of ethanol, methanol, isopropanol and water.

[0033] In some embodiments of the present invention, the mass ratio of dodecylamine, ethyl silicate, triethyl phosphate and calcium nitrate tetrahydrate is 30-50 g: 150-180 mL: 100-110 mL: 240-250 mL.

[0034] In some embodiments of the present invention, the mixing reaction time is 12 to 48 hours.

[0035] In some embodiments of the present invention, the washing solvent is water and / or ethanol.

[0036] In some embodiments of the present invention, the content of the bioactive glass in the cartilage induction solution is 0.05-10 mg / mL.

[0037] In some preferred embodiments of the present invention, the content of the bioactive glass in the cartilage induction solution is 0.05-0.5 mg / mL.

[0038] In some embodiments of the present invention, the conditions for continued culture are 37±2°C and 4%~6% CO2.

[0039] In some embodiments of the present invention, the continued culturing time is 5 to 10 days.

[0040] In some preferred embodiments of the present invention, the continued culture time is 6 to 8 days.

[0041] In some embodiments of the present invention, half of the medium is replaced every 1 to 2 days during the continued culture.

[0042] In some embodiments of the present invention, the callus organoid construction is carried out in a low adhesion well plate.

[0043] In some embodiments of the present invention, the low-adhesion well plate is a well plate coated with 1-3% agarose, preferably a 6-well plate.

[0044] A second aspect of the present invention provides a callus organoid obtained by the callus organoid construction method described in any one of the first aspects.

[0045] The third aspect of the present invention provides the method for constructing callus organoids as described in any one of the first aspect or the use of callus organoids as described in the second aspect in preparing bone defect repair products or drug screening.

[0046] Other features and advantages of the present invention will be set forth in the description which follows. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] The present invention will be further described below with reference to the accompanying drawings and embodiments, wherein: Figure 1 This is an optical microscope observation image of the hydrogel microspheres of the embodiment of the present invention; Figure 2 This is an electron microscope image of the bioactive glass of an embodiment of the present invention; Figure 3 This is an Alcian blue staining image of the callus organoid based on endochondral ossification of the present invention; Figure 4 These are light microscopic photographs of callus organoids constructed based on the endochondral ossification process of the present invention, wherein A is the callus organoid constructed in Comparative Example 1, and B is the callus organoid constructed in Example 1; Figure 5 This is a diagram showing the alkaline phosphatase staining results of the callus organoids based on the endochondral ossification process constructed in the present invention, wherein A is the callus organoid constructed in Comparative Example 1, and B is the callus organoid constructed in Example 1; Figure 6 This is an Alizarin red staining result of the callus organoid constructed based on the endochondral ossification process of the present invention, wherein A is the callus organoid constructed in Comparative Example 1, and B is the callus organoid constructed in Example 1; Figure 7 This is a graph showing the immunofluorescence staining results of the callus organoid based on the endochondral ossification process constructed in the present invention, wherein A is the Sox9 protein detection result, B is the Col X protein detection result, C is the Runx2 protein detection result, and D is the ALP protein detection result. DETAILED DESCRIPTION

[0048] The following will be combined with the embodiments to clearly and completely describe the concept of the present invention and the technical effects produced, so as to fully understand the purpose, characteristics and effects of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without creative work are all within the scope of protection of the present invention.

[0049] The words "preferably", "more preferably", etc. in the present invention refer to embodiments of the present invention that may provide certain beneficial effects in certain circumstances. However, other embodiments may also be preferred under the same circumstances or other circumstances. In addition, the description of one or more preferred embodiments does not imply that other embodiments are not applicable, nor is it intended to exclude other embodiments from the scope of the present invention.

[0050] When a numerical range is disclosed herein, the above range is considered to be continuous and includes the minimum and maximum values ​​of the range, as well as every value between such minimum and maximum values. Further, when a range refers to an integer, every integer between the minimum and maximum values ​​of the range is included. In addition, when multiple ranges are provided to describe features or characteristics, the ranges can be combined. In other words, unless otherwise indicated, all ranges disclosed herein should be understood to include any and all subranges included therein.

[0051] In the description of the present invention, the reference term "and / or" includes all and any combinations of one or more of the associated listed items.

[0052] In the description of the present invention, the description with reference to the terms "one embodiment", "some embodiments", "illustrative embodiments", "examples", "specific examples", or "some examples" means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representation of the above terms does not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner.

[0053] If the specific conditions are not specified in the examples, the experiments were carried out under conventional conditions or conditions recommended by the manufacturer. If the manufacturers of the reagents or instruments are not specified, they are all conventional products that can be purchased commercially.

[0054] Example 1: Method for constructing callus organoids based on endochondral ossification The present embodiment provides a method for constructing callus organoids based on endochondral ossification, which is to inoculate stem cells into hydrogel microspheres constructed by extracellular matrix or its derivatives, culture and construct chondrocytes, and then induce cartilage hypertrophy and osteogenesis to construct callus organoids, which specifically includes the following contents.

[0055] 1. Preparation of hydrogel microspheres 1 g of gelatin methacrylate (GelMA, purchased from Aladdin) was dispersed in 5 g of deionized water, heated in a 40°C water bath to dissolve, and then 0.25% of phenyl (2,4,6-trimethylbenzoyl) lithium phosphate was added to prepare solution A.

[0056] Solution B was prepared by dissolving 3 g of sorbitan oleate (Span 80) in 100 mL of isopropyl acetate.

[0057] Then, a syringe pump was used to drop solution A into solution B at a rate of 200 μL / min. During the dropwise addition, magnetic stirring was continuously performed to obtain hydrogel microspheres. Finally, the active agent on the surface of the hydrogel microspheres was washed off with alcohol and deionized water, and the hydrogel microspheres were collected, absorbed, and freeze-dried to obtain the final GelMA hydrogel microspheres for later use.

[0058] The results of optical microscopy observation of the hydrogel microspheres prepared by this method are as follows: Figure 1 As shown, its diameter is about 400~500μm.

[0059] 2. Preparation of bioactive glass First, adjust the water bath temperature to 40°C, dissolve 40 g of dodecylamine (DDA) in 250 ml of deionized water and 800 ml of anhydrous ethanol in a water bath, stir for 10 min, then add ethyl silicate (TEOS, 160 ml), triethyl phosphate (TEP, 104.9 ml) and calcium nitrate tetrahydrate (CN, 242.1 g) to the solution in sequence, stir for 30 min after adding each reagent, then slowly add the next one. Continue stirring for 3 h and age for 24 h to obtain the precursor.

[0060] The precursor obtained above was centrifuged, washed alternately with deionized water and anhydrous ethanol for 3 times, and finally placed in a muffle furnace and heat treated at 650° C. for 3 h to obtain bioactive glass (BG) for later use.

[0061] The electron microscope observation picture of the bioactive glass prepared by this method is as follows Figure 2 As shown, the particle size is about 200~500 nm.

[0062] 3. Construction of microsphere-cell complex A 6-well plate was coated with 2% agarose to form a low-adhesion well plate condition, and then the attached bone marrow mesenchymal stem cells were digested, and then 40 mg of the above hydrogel microspheres (GelMA) were added and the density of 5×10 5 The cells were uniformly seeded in the above low-adhesion 6-well plates with a diameter of 400-500 μm. Then they were placed in a 37±0.2℃, 5% CO2 incubator for static culture, and half of the medium was replaced with complete medium every other day.

[0063] The complete culture medium was DMEM high-glucose medium (Gibco) supplemented with 10% FBS and 1% double antibody.

[0064] 4. Constructing a cartilage ball When the surface growth density of the bone marrow mesenchymal stem cells on the GelMA hydrogel microspheres reached 70%, the cartilage induction medium was replaced and cultured. The culture conditions were 37±0.2℃, 5% CO2, and the medium was half changed every other day. After 14 days of culture, chondrocytes were obtained. The chondrocytes were stained with Alcian blue. The results were as follows: Figure 3 As shown, after 14 days of chondrogenic induction, the cell-loaded hydrogel microspheres secreted a large amount of cartilage matrix and chondrogenic spheres were successfully constructed.

[0065] The chondrogenic induction medium contained 10% fetal bovine serum (FBS), 1% double antibody (penicillin-streptomycin, Sciencell, USA), 100 nM dexamethasone, 0.2 mM L-ascorbic acid-2-phosphate trisodium salt, 40 μg / mL proline, 1 mM sodium pyruvate, 1% ITS (Gibco), 10 ng / ml TGFβ3 and DMEM high glucose medium.

[0066] 5. Construction of callus organoids On the 14th day of cartilage induction, the chondrocytes were placed in a cartilage induction solution containing 0.1 mg / ml of the bioactive glass and cultured for 7 days to obtain callus organoids based on the endochondral ossification process.

[0067] Comparative Example 1: No bioactive glass This comparative example provides a method for constructing callus organoids, which differs from Example 1 in that no bioactive glass is added during the construction of callus organoids, and the remaining steps are the same.

[0068] The method for constructing callus organoids in this comparative example is as follows: Chondrocytes were obtained by referring to the method of Example 1 above, and then on the 14th day of chondrogenic induction, the chondrocytes were cultured in the chondrogenic induction medium for 7 days, with half of the medium being changed every other day to obtain callus organoids.

[0069] Test example This test example detects and characterizes the morphology and molecular characteristics of the callus organoids constructed in Example 1 and Comparative Example 1 above, as follows: 1. Optical microscope observation The callus organoids constructed in Example 1 and Comparative Example 1 were observed using an optical microscope, and the optical microscope photos thereof are as follows: Figure 4 As shown, Figure 4 A in the figure is the callus organoid constructed in comparative example 1 (i.e., only cartilage induction medium was added during cartilage hypertrophy and osteogenesis). Figure 4B in the figure is the callus organoid constructed in Example 1 of the present invention (i.e., cartilage induction medium and 0.1 mg / mL bioactive glass are added during cartilage hypertrophy and osteogenesis). The results show that the bioactive glass is evenly dispersed on the surface of the constructed callus organoid, providing it with good osteogenesis and self-mineralization ability subsequently.

[0070] 2. Alkaline phosphatase detection First, wash the callus organoids with PBS and fix them with 4% (w / v) paraformaldehyde (incubate at room temperature for 30 minutes). After fixation, wash them with PBS three times, 5 minutes each time. Mix the corresponding detection reagents in the alkaline phosphatase colorimetric kit (Biyuntian, C3206) in proportion to prepare BCIP / NBT staining working solution. After the last wash, remove PBS and add an appropriate amount of BCIP / NBT staining working solution to ensure that the sample is fully covered. Incubate at room temperature in the dark for 5-30 minutes until the color develops to the expected depth. Remove the BCIP / NBT staining working solution and wash with distilled water 1-2 times to terminate the color development reaction.

[0071] Alkaline phosphatase staining results Figure 5 As shown, Figure 5 A in the figure is the callus organoid constructed in Comparative Example 1, Figure 5 B in the figure is the callus organoid constructed in Example 1 of the present invention. The results show that the callus organoid constructed by the method of the present invention secretes more alkaline phosphatase than the callus organoid constructed in Comparative Example 1.

[0072] 3. Detection of calcium salt deposition The callus organoids were first washed with PBS and fixed with 4% (w / v) paraformaldehyde (incubated at room temperature for 30 minutes). After fixation, they were washed with PBS three times, 5 minutes each time. Then they were stained with Alizarin Red for 30 minutes. After staining, they were washed with PBS until the PBS no longer changed color. The calcium salt deposition in the callus organoids was observed under a microscope (Olympus, Tokyo).

[0073] Alizarin red staining results Figure 6 As shown, Figure 6 A in the figure is the callus organoid constructed in Comparative Example 1, Figure 6 B is the callus organoid constructed in Example 1 of the present invention. The results show that the callus organoid constructed by the method of the present invention forms more calcium salt deposits and mineralized nodules than the callus organoid constructed in Comparative Example 1.

[0074] 4. Immunofluorescence detection The callus organoids were first washed with PBS and then fixed with 4% (w / v) paraformaldehyde (incubated at room temperature for 30 minutes). After fixation, they were washed three times with PBS for 5 minutes each time. They were blocked with 5% bovine serum albumin for 1 hour at room temperature. The samples were then incubated with primary antibodies overnight at 4 °C. After washing with PBS, the samples were incubated with fluorescein-conjugated secondary antibodies for 1.5 hours, and cell nuclei were stained with DAPI (Solarbio) for 10 minutes. The stained samples were observed under a confocal microscope system (Leica), and all immunofluorescence images were obtained as single images using the Z-stack function.

[0075] The primary antibodies used were: Sox9 (proteintech, 67439-1-Ig), Col X (ABclonal, A11645), Runx2 (proteintech, 20700-1-AP), and Alp (Affinit, DF6225).

[0076] Immunofluorescence test results Figure 7 As shown, A is the result of Sox9 protein detection, B is the result of Col X protein detection, C is the result of Runx2 protein detection, and D is the result of ALP protein detection. The results show that the expression of cartilage tissue index (Sox9 protein) of the callus organoid constructed by the embodiment of the present invention is lower than that of the comparative example 1, while the expression of chondrocyte hypertrophy index (Col X protein) and early osteogenesis index (Runx2, ALP protein) are significantly better than that of the comparative example 1. It can be seen that the callus organoid constructed by the method of the embodiment of the present invention has been transformed from chondrocytes to hypertrophic osteogenesis faster than the comparative example, and has excellent osteogenic differentiation and matrix mineralization ability.

[0077] In summary, the present invention provides a method and application for constructing callus organoids based on endochondral ossification. The present invention utilizes stem cells to sequentially induce the generation of self-mineralized callus organoids in vitro, which have good osteogenic differentiation and matrix mineralization capabilities and can be used for bone defect repair.

[0078] The above is a detailed description of the embodiments of the present invention, but the present invention is not limited to the above embodiments. Various changes can be made within the knowledge of ordinary technicians in the relevant technical field without departing from the purpose of the present invention. In addition, the embodiments of the present invention and the features in the embodiments can be combined with each other without conflict.

Claims

1. A method for constructing callus organoids based on endochondral ossification, characterized in that: The following steps are involved: The hydrogel microspheres loaded with stem cells are placed in a cartilage induction solution for culture to obtain cartilage spheres; and then the cartilage spheres are placed in the cartilage induction solution containing bioactive glass for further culture to obtain the product.

2. The method for constructing callus organoids according to claim 1, characterized in that: The preparation method of the hydrogel microspheres loaded with stem cells comprises: In complete culture medium, mix stem cells and hydrogel microspheres and culture until the surface growth density reaches 60%~80%.

3. The method for constructing callus organoids according to claim 2, characterized in that: The complete culture medium contains 8%-12% FBS, 0.5%-1.5% double antibody and DMEM high glucose culture medium; and / or, the stem cells include totipotent stem cells or pluripotent stem cells; And / or, the mixed culture conditions are 37±2°C, 4%~6% CO2; And / or, the mixed culture time is 5 to 14 days.

4. The method for constructing callus organoids according to claim 1, characterized in that: The hydrogel microspheres include at least one of methacrylated gelatin hydrogel microspheres, hyaluronic acid hydrogel microspheres, chitosan hydrogel microspheres, sodium alginate hydrogel microspheres, and gelatin hydrogel microspheres.

5. The method for constructing callus organoids according to claim 4, characterized in that: The diameter of the hydrogel microspheres is 100-1000 μm.

6. The method for constructing callus organoids according to claim 1, characterized in that: The cartilage induction solution comprises 8% to 12% fetal bovine serum, 0.5% to 1.5% penicillin-streptomycin double antibody, 80 to 120 nM dexamethasone, 0.1 to 0.3 mM L-ascorbic acid-2-phosphate trisodium salt, 30 to 50 μg / mL proline, 0.8 to 1.2 mM sodium pyruvate, 0.8% to 1.2% ITS, 8 to 12 ng / ml TGFβ3 and DMEM high glucose medium; And / or, the culturing time is 12 to 18 days.

7. The method for constructing callus organoids according to claim 1, characterized in that: The preparation method of the bioactive glass comprises: Under solvent conditions, dodecylamine, ethyl silicate, triethyl phosphate and calcium nitrate tetrahydrate are added, mixed and reacted to obtain a precursor, which is then centrifuged, washed and calcined at 600-700°C for 2.5-3.5 h to obtain a precursor; Preferably, the content of the bioactive glass in the cartilage induction solution is 0.05-10 mg / mL.

8. The method for constructing callus organoids according to claim 1, characterized in that: The time for continuing cultivation is 5 to 10 days.

9. A callus organoid, characterized in that: Obtained by the callus organoid construction method according to any one of claims 1 to 8.

10. Use of the method for constructing callus organoids according to any one of claims 1 to 8 or the callus organoids according to claim 9 in preparing bone defect repair products or drug screening.

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