Bone organoid and its construction method and use

Abstract

The present invention belongs to the field of biomedical engineering technology and relates to a bone organoid, its construction method, and its use. The bone organoid is obtained by three-dimensionally culturing a cell mixture comprising stem cells and macrophages. The bone organoid and its construction method of the present invention enable rapid preparation of bone organoids with minimal differences from natural bone, enabling regulation of cell fate and better bone formation and repair.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedical engineering and relates to a bone organoid and a construction method and use thereof. Background Art

[0002] Bone defects in the oral, craniomaxillofacial, and other parts of the body have diverse causes, high incidence rates, complex morphologies, and difficulty in bone regeneration, which seriously affect the patient's appearance, chewing, pronunciation, and movement functions, leading to severe damage to the patient's quality of life and physical and mental health. Currently, small bone defects can be repaired by filling with materials such as artificial bone powder. For larger bone defects, current treatment methods include autologous bone transplantation, allogeneic transplantation, and synthetic bone substitutes, but these methods have limitations. Autologous bone transplantation causes damage and severe trauma to the donor site; allogeneic transplantation and synthetic bone substitutes have problems such as immunogenicity, infection, poor integration, and insufficient vascularization.

[0003] Organoids are simple, cell-based in vitro models for tissue engineering. They are three-dimensional cultures with biomimetic spatial characteristics formed by cells through self-renewal and self-organization, or through the induction of biomaterials. They can simulate or partially simulate the cell types, structures, and functions of tissues and organs in the body. Currently, organoids play a vital role in the study of tissue development, disease occurrence and development mechanisms, drug screening, and tissue engineering. Therefore, engineered tissues constructed based on bone organoids can provide a new approach for bone tissue regeneration and repair, and can also serve as a model for studying bone tissue development and drug screening.

[0004] However, in bone organoid technology, despite researchers' extensive efforts in the construction and application of bone organoids, the construction of bone organoids is time-consuming, lacks the regulation of cell fate, has poor osteogenesis effects, and has a single cell composition that differs significantly from natural bone. For example, the patent application "A 3D bioprinted bone tissue engineering scaffold based on bone organoids" (application number: CN202210911808.X) constructs a bone organoid comprising a shell, an outer biomimetic periosteum, and an internal microsphere filling. It simulates the macrostructure of bone tissue in a static environment, but does not simulate the dynamic mechanical microenvironment that is indispensable in the process of bone tissue formation, and lacks the regulation of cell fate. Another example is the patent application "A system for constructing bone organoids and its use method" (application number: CN202311219797.X), which selects at least one cell or multiple cells including bone marrow stromal cells, osteoprogenitor cells, preosteoblasts, osteoblasts, bone lining cells, osteocytes, or osteoclasts to construct bone organoids. For example, the paper "Developmentally Engineered Callus Organoid Bioassemblies Exhibit Predictive In Vivo Long Bone Healing" (DOI number: 10.1002 / advs.201902295; PMID number: 31993293) chose to construct bone organoids using stem cells from a single periosteum. However, these cells are mostly osteoblasts and lack immune cells such as macrophages that play a key role in bone immune homeostasis and bone repair and regeneration. Summary of the Invention

[0005] The primary purpose of the present invention is to provide a bone organoid that can be rapidly constructed, has little difference from natural bone, can regulate cell fate, and can better promote bone formation and bone repair.

[0006] To achieve this object, in a basic embodiment, the present invention provides a bone organoid, wherein the bone organoid is obtained by three-dimensionally culturing a cell mixture, wherein the cell mixture comprises stem cells and macrophages.

[0007] In a preferred embodiment, the present invention provides a bone organoid, wherein the stem cells are selected from one or more of umbilical cord blood mesenchymal stem cells, bone marrow mesenchymal stem cells, adipose mesenchymal stem cells, and induced pluripotent stem cells.

[0008] In a preferred embodiment, the present invention provides a bone organoid, wherein the macrophages are bone marrow-derived macrophages and / or peripheral blood-derived macrophages.

[0009] In a preferred embodiment, the present invention provides a bone organoid, wherein the ratio of the number of the stem cells to the number of the macrophages is (2-3):1.

[0010] In a preferred embodiment, the present invention provides a bone organoid, wherein the cell mixture further comprises a hydrogel matrix material.

[0011] In a preferred embodiment, the present invention provides a bone organoid, wherein the hydrogel matrix material is selected from one or more of methacrylated gelatin, hyaluronic acid, sodium alginate, silk fibroin, carboxymethyl chitosan, chondroitin sulfate, and collagen.

[0012] In a preferred embodiment, the present invention provides a bone organoid, wherein the density of all cells formed in the hydrogel matrix material is (1-5)×10 6 pieces / mL.

[0013] The second object of the present invention is to provide a method for constructing bone organoids as described above, so as to enable rapid preparation of bone organoids as described above, with the bone organoids having little difference from natural bone, being able to regulate cell fate, and being able to better promote bone formation and bone repair.

[0014] To achieve this objective, in a basic embodiment, the present invention provides a method for constructing a bone organoid as described above, the method comprising the following steps:

[0015] (1) uniformly mixing the materials in the cell mixture in a three-dimensional culture reactor;

[0016] (2) solidifying the hydrogel matrix material;

[0017] (3) Performing cell culture to obtain the bone organoid.

[0018] In a preferred embodiment, the present invention provides a method for constructing bone organoids as described above, wherein in step (1), the three-dimensional culture reactor is a three-dimensional culture reactor based on dynamic tensile stress.

[0019] Three-dimensional culture is a technique for cell growth, proliferation, and migration in three dimensions. Cells are encapsulated in a three-dimensional matrix or a synthetic scaffold. Compared to traditional two-dimensional cell culture, 3D cell culture more closely resembles the physiological state of cells in vivo, including cell-cell interactions, signaling, and responses to the extracellular matrix. Therefore, 3D culture is more capable of mimicking the biological properties of tissues and organs, offering greater potential for biomedical research, drug development, and tissue engineering. For a detailed introduction to 3D culture, see the article: Functional and Biomimetic Materials for Engineering of the Three-Dimensional Cell Microenvironment (DOI: 10.1021 / acs.chemrev.7b00094).

[0020] Dynamic tensile stress three-dimensional culture is different from the previous three-dimensional culture in a static environment. It is a three-dimensional culture method based on a dynamic environment. It combines the entire three-dimensional culture system with the chamber, and uses dynamic tensile stress equipment (such as CELL TANK equipment) to dynamically stretch the chamber, thereby driving the dynamic stretching of the entire three-dimensional culture system, so that dynamic tensile stress is generated at the same time as the stretching deformation. Therefore, the cells in culture are also in a dynamic tensile stress environment.

[0021] In a preferred embodiment, the present invention provides a method for constructing bone organoids as described above, wherein the three-dimensional culture reactor based on dynamic tensile stress is subjected to plasma spraying treatment to remove impurities on the inner surface and polylysine coating on the inner surface to improve the hydrophilicity and adhesion of the inner surface before adding the cell mixture.

[0022] In a preferred embodiment, the present invention provides a method for constructing bone organoids as described above, wherein in step (2), the curing is photocuring, the wavelength of the photocuring is 400-420 nm, and the photocuring time is 30-60 s.

[0023] In a preferred embodiment, the present invention provides a method for constructing bone organoids as described above, wherein in step (3), during cell culture, the frequency of dynamic tensile stress is 0.5-0.8 Hz, the time is 2-3 h / day, and the ratio is 8-10%.

[0024] The third object of the present invention is to provide the use of the bone organoids described above for preparing bone repair materials or in vitro research models, so as to better promote osteogenesis and bone repair.

[0025] To achieve this object, in a basic embodiment, the present invention provides the use of the bone organoid as described above for preparing a bone repair material or an in vitro research model.

[0026] In a preferred embodiment, the present invention provides the use of the bone organoids described above for preparing bone repair materials or in vitro research models, wherein the bone repair materials are used to treat bone defects or osteoporosis, and the in vitro research models are used for tissue development research or drug screening research.

[0027] The beneficial effect of the present invention is that the bone organoids and the construction method thereof can be used to quickly prepare bone organoids, and the prepared bone organoids have little difference from natural bones, can regulate cell fate, and can better promote bone formation and bone repair.

[0028] Currently, bone organoids are mostly constructed using single osteoblasts without considering the bone immune microenvironment, which is significantly different from natural bone tissue. The present invention introduces immune cells such as macrophages on the basis of osteoblasts to regulate the bone immune microenvironment, thereby forming functional bone organoids with immune regulatory effects.

[0029] Currently, the construction of bone organoids is time-consuming, lacks regulation of cell fate, and has poor osteogenesis effect. The present invention simulates the mechanical environment of natural bone tissue, constructs a dynamic tensile stress three-dimensional culture system, and mechanically regulates cell fate. At the same time, it can shorten the time of bone organoid construction. Obtaining bone organoids can improve the osteogenesis effect and promote the formation of mature functional bone organoids.

[0030] The bone organoids provided by the present invention have significantly improved abilities to promote osteogenic differentiation of mesenchymal stem cells and inhibit bone loss compared with mesenchymal stem cell apoptosis vesicles alone, and have broad clinical application prospects in the treatment of senile osteoporosis.

[0031] Compared to previous studies of bone organoids based on static culture and single-cell construction, the bone organoids constructed in this paper demonstrate superior bone repair efficacy. Currently, autologous bone transplantation is the gold standard for treating bone defects, but autologous bone transplantation can cause damage to the donor site. The bone organoids constructed in this paper achieve bone regeneration and repair comparable to autologous bone repair just 14 days after implantation. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 Flowchart showing an exemplary method for constructing bone organoids according to the present invention.

[0033] Figure 2 These are graphs showing the alkaline phosphatase (ALP) assay results for the bone organoids constructed in Examples 1-3 and Comparative Examples 1-8, including: (a) ALP staining, scale bar: 1 cm; (b) ALP activity quantification.

[0034] Figure 3 These are the detection results of immune polarization of bone organoids constructed in Comparative Example 6 and Example 1, including: (a) expression of immune polarization-related genes (**P < 0.01, ***P < 0.001); (b) immunofluorescence staining of the immune polarization-related protein iNOS; (c) immunofluorescence staining of the immune polarization-related protein ARG-1.

[0035] Figure 4 Graphs showing the femoral defect repair results 14 days after implantation of bone organoids constructed in Example 1, Comparative Example 1, Comparative Example 6, and Comparative Example 8, including: (a) Micro-CT scan reconstruction and (b) quantitative analysis (ns, no statistical difference, *P < 0.05, **P < 0.01, ***P < 0.001, scale bar: 1 mm). DETAILED DESCRIPTION

[0036] The specific embodiments of the present invention are further described below through examples and drawings.

[0037] Example 1: Cultivation of bone organoids (I)

[0038] according to Figure 1 The process shown in the figure is used to culture bone organoids, which specifically includes the following steps:

[0039] (1) The inner surface of the dynamic tensile stress chamber (CELL TANK, 20×20 mm) was first plasma sprayed with nitrogen (the plasma nozzle was 2 mm away from the inner surface and the movement speed was 2 mm / s) to remove impurities on the inner surface. Then, a layer of poly-lysine solution (0.1% (W / V)) was coated on the inner surface and incubated overnight in the dark. The poly-lysine solution was discarded the next day and dried at 60°C for 1 h before use to improve the hydrophilicity and adhesion of the chamber surface.

[0040] (2) 10 wt% (5-15 wt%) methacryloyl gelatin (GelMA) was mixed with bone marrow mesenchymal stem cells (obtained by adherence screening method: flush out the cells in the bone marrow cavity with complete culture medium (α-MEM medium + 10% (mass fraction) fetal bovine serum + 1% (mass fraction) penicillin-streptomycin) using a syringe, collect the bone marrow fluid and inoculate it into a culture dish, remove the suspended cells after 48 hours, and the adherent cells are bone marrow mesenchymal stem cells), bone marrow-derived macrophages (obtained by adherence screening + macrophage colony stimulating factor induction method) The cells in the bone marrow cavity were flushed out with complete culture medium (α-MEM medium + 10% (mass fraction) fetal bovine serum + 1% (mass fraction) penicillin-streptomycin) using a syringe, and the bone marrow fluid was collected and inoculated into a culture dish. After 48 hours, the suspended cells were collected and re-inoculated into the culture dish. Macrophage colony-stimulating factor was added at a working concentration of 40 ng / ml. After 72 hours of culture, the adherent cells were bone marrow-derived macrophages. The ratio of bone marrow mesenchymal stem cells to bone marrow-derived macrophages was 2:1, and the final cell density was 3×10 6 The mixture was then injected into a dynamic tensile stress chamber and cured with 405 nm light for 60 seconds, resulting in the hydrogel adhering tightly to the dynamic tensile stress chamber.

[0041] (3) The chamber was placed in a dynamic tensile stress device (CELL TANK, corresponding to 20×20 mm), and the proportion, frequency, and time of dynamic tensile stress were set to 10%, 0.5 Hz, and 2 h / day, respectively, so that the dynamic tensile stress chamber was deformed and the three-dimensional culture system was driven to undergo dynamic tensile stress. Cell culture was started (cultured in a 37°C constant temperature incubator containing 5% (volume fraction) CO2 + 95% (volume fraction) air; complete culture medium composition: α-MEM medium + 10% (mass fraction) fetal bovine serum + 1% (mass fraction) penicillin-streptomycin; the culture medium was changed every 2 days) for a total of 28 days.

[0042] Example 2: Cultivation of Bone Organoids (II)

[0043] The ratio of bone marrow mesenchymal stem cells to bone marrow-derived macrophages in step (2) was changed to 3:1.

[0044] Other details are the same as in Example 1.

[0045] Example 3: Cultivation of bone organoids (III)

[0046] The proportion, frequency and time of dynamic tensile stress in step (3) were changed to 8%, 0.8 Hz and 3 h / day respectively.

[0047] Other details are the same as in Example 1.

[0048] Comparative Example 1: Culture of Bone Organoids (IV)

[0049] In step (2), only the same amount of bone marrow mesenchymal stem cells were added without adding bone marrow-derived macrophages, and the final cell density changed accordingly.

[0050] Other details are the same as in Example 1.

[0051] Comparative Example 2: Culture of Bone Organoids (V)

[0052] The ratio of bone marrow mesenchymal stem cells to bone marrow-derived macrophages in step (2) was changed to 1:1.

[0053] Other details are the same as in Example 1.

[0054] Comparative Example 3: Culture of Bone Organoids (VI)

[0055] The ratio of bone marrow mesenchymal stem cells to bone marrow-derived macrophages in step (2) was changed to 4:1.

[0056] Other details are the same as in Example 1.

[0057] Comparative Example 4: Culture of Bone Organoids (VII)

[0058] The proportion, frequency and time of dynamic tensile stress in step (3) were changed to 15%, 0.25 Hz and 1 h / day respectively.

[0059] Other details are the same as in Example 1.

[0060] Comparative Example 5: Culture of Bone Organoids (VIII)

[0061] The proportion, frequency and time of dynamic tensile stress in step (3) were changed to 5%, 1 Hz and 4 h / day respectively.

[0062] Other details are the same as in Example 1.

[0063] Comparative Example 6: Culture of Bone Organoids (IX)

[0064] In step (3), the ratio, frequency and time of dynamic tensile stress are not set, that is, dynamic tensile stress three-dimensional culture is not performed, but static culture is performed.

[0065] Other details are the same as in Example 1.

[0066] Comparative Example 7: Culture of Bone Organoids (X)

[0067] The bone marrow-derived macrophages in step (2) are replaced with osteoclasts.

[0068] Other details are the same as in Example 1.

[0069] Comparative Example 8: Culture of Bone Organoids (XI)

[0070] In step (2), only the same amount of bone marrow mesenchymal stem cells were added without adding bone marrow-derived macrophages, and the final cell density changed accordingly.

[0071] In step (3), the ratio, frequency and time of dynamic tensile stress are not set, that is, dynamic tensile stress three-dimensional culture is not performed, but static culture is performed.

[0072] Other details are the same as in Example 1.

[0073] Example 4: Identification of Bone Organoids

[0074] The bone organoids prepared in Examples 1-3 and Comparative Examples 1-7 were identified as follows.

[0075] Alkaline phosphatase (ALP) staining kit (Beyotime, China) and ALP quantification kit (Beyotime, China) were used for ALP staining and ALP activity quantification. Figure 2 .Depend on Figure 2 The results show that the ALP staining of the bone organoids constructed in Examples 1-3 is darker than that in Comparative Examples 1-8. ALP quantification also shows that the ALP activity of the bone organoids constructed in Examples 1-3 is higher than that in Comparative Examples 1-8, and the osteogenic differentiation is better.

[0076] This shows that compared with using bone marrow mesenchymal stem cells alone, the addition of bone marrow-derived macrophages can better construct bone organoids, better regulate cell fate, and better osteogenic differentiation; compared with bone marrow mesenchymal stem cells plus osteoclasts, bone marrow mesenchymal stem cells plus bone marrow-derived macrophages can better construct bone organoids, better regulate cell fate, and better osteogenic differentiation; compared with static culture, dynamic tensile stress culture can better construct bone organoids, better regulate cell fate, and better osteogenic differentiation; in dynamic tensile stress culture, the optimal culture conditions are a ratio of 2-3:1 between bone marrow mesenchymal stem cells and bone marrow-derived macrophages, a frequency of 0.5-0.8 Hz, a time of 2-3 h / day, and a ratio of 8-10%.

[0077] Real-time fluorescence quantitative PCR (qPCR) was used to detect the expression of macrophage polarization-related genes in bone organoids to observe whether dynamic tensile stress would regulate immune polarization in bone organoids. Figure 3 As shown in the figure, dynamic tensile stress culture reduced the expression of pro-inflammatory factors Tnf-α and Il-1β, and increased the expression of anti-inflammatory factors Tgf-β and Il-10.

[0078] By performing immunofluorescence staining on the marker proteins of macrophage immune polarization in bone organoids, we observed whether dynamic tensile stress would regulate the immune polarization in bone organoids. Figure 3 As shown in the figure, dynamic tensile stress culture reduced the expression of pro-inflammatory marker iNOS and increased the expression of anti-inflammatory marker ARG-1.

[0079] In summary, dynamic tensile stress culture and the addition of macrophages can regulate the immune microenvironment within bone organoids and promote the transformation of bone organoids to an anti-inflammatory immune microenvironment that is more conducive to bone tissue repair.

[0080] Example 5: Experiment on repairing femoral defects in mice using bone organoids

[0081] The bone organoids prepared in Examples 1-3, Comparative Example 1, Comparative Example 6, and Comparative Example 8 and autologous bone (taken from the contralateral tibia of the femoral defect side of the mouse) were tested for repairing femoral defects in mice. The specific test methods were as follows:

[0082] After 21 days of in vitro culture, bone organoids and autologous bone were implanted into femoral defects (1 mm diameter) in mice (C57BL / 6N, Department of Animal Science, Peking University Health Science Center, n=4 per group). Samples were harvested 14 days postoperatively and fixed in 4% paraformaldehyde for 24 hours before micro-CT scanning and 3D reconstruction for analysis. The following methods were used: a 0.6 cm incision was made on the lateral surface of the distal femur of the mouse hind limb with a scalpel, and the muscle and periosteum were separated to completely expose the distal femur. A 1 mm diameter perforated defect was created at the distal femur with a ball drill, and the bone organoid repair material was implanted. The muscle was sutured with absorbable sutures, and the skin was sutured with non-absorbable sutures. The wound was disinfected with iodine swabs, and 10,000 penicillin was injected intramuscularly in the hind limbs for infection. After 14 days, the mice were killed by cervical dislocation for sample collection. The femoral samples were fixed in 4% paraformaldehyde for 2 days and then scanned with high-resolution micro-CT. The scans were visualized using the accompanying 3D visualization software Inveon Research. Three-dimensional reconstruction of the tomographic images of the femoral defect site of mice was performed at workplace to further quantitatively analyze bone mineral density (BMD), bone volume / total volume ratio (BV / TV), trabecular thickness (Tb.Th) and trabecular number (Tb.N).

[0083] The results are as follows Figure 4 As shown, we can see:

[0084] (1) The static cultured organoids of bone marrow mesenchymal stem cells (BMSCs) + bone marrow-derived macrophages (BMDMs) (Comparative Example 6) have better bone repair effects than the static cultured bone organoids of BMSCs (Comparative Example 8); the dynamic tensile stress bone organoids of BMSCs + BMDMs (Example 1) have better bone repair effects than the dynamic tensile stress bone organoids of BMSCs (Comparative Example 1), indicating that the addition of macrophages can better repair bone defects.

[0085] (2) BMSCs dynamic tensile stress bone organoids (Comparative Example 1) have better bone repair effects than BMSCs static culture bone organoids (Comparative Example 8); BMSCs+BMDMs dynamic tensile stress bone organoids (Example 1) have better bone repair effects than BMSCs+BMDMs static culture bone organoids (Comparative Example 6), indicating that bone organoids constructed based on dynamic tensile stress can better repair bone defects than static culture bone organoids.

[0086] (3) The repair effect of BMSCs+BMDMs dynamic tensile stress bone organoids (Example 1) was the best. After 14 days of implantation, the bone defect was almost filled with new trabeculae, which could achieve a repair effect comparable to that of autologous bone.

[0087] As can be seen from the above examples:

[0088] (1) When constructing bone organoids, the present invention regulates cell fate through stress and regulates the bone immune microenvironment by introducing macrophages, which significantly accelerates the formation speed and quality of functional bone organoids.

[0089] (2) The bone organoids constructed by the present invention have a good bone defect repair effect. 14 days after implantation, they have a repair effect comparable to the gold standard of treatment - autologous bone transplantation.

[0090] (3) The bone organoids constructed by the present invention can also be used as a model for studying bone tissue development and a drug screening model.

[0091] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these changes and variations. The above embodiments or implementation methods are merely illustrative of the present invention, and the present invention may also be implemented in other specific ways or other specific forms without departing from the gist or essential features of the present invention. Therefore, the described implementation methods should be regarded as illustrative and not restrictive in any respect. The scope of the present invention should be described by the appended claims, and any changes that are equivalent to the intent and scope of the claims should also be included within the scope of the present invention.

Claims

1. A bone organoid, characterized in that: The bone organoids are obtained by three-dimensional culture of a cell mixture, wherein the cell mixture comprises stem cells, macrophages and a hydrogel matrix material, and the ratio of the number of the stem cells to the number of the macrophages is (2-3):

1. The stem cells are bone marrow mesenchymal stem cells, and the macrophages are bone marrow-derived macrophages. The three-dimensional culture step includes uniformly mixing the materials in the cell mixture and solidifying the hydrogel matrix material, and then performing cell culture based on dynamic tensile stress.

2. The bone organoid according to claim 1, wherein: The hydrogel matrix material is selected from one or more of methacrylated gelatin, hyaluronic acid, sodium alginate, silk fibroin, carboxymethyl chitosan, chondroitin sulfate, and collagen; The density of all cells formed in the hydrogel matrix material is (1-5)×10 6 pieces / mL.

3. A method for constructing bone organoids according to claim 1 or 2, characterized in that: The construction method comprises the following steps: (1) uniformly mixing the materials in the cell mixture in a three-dimensional culture reactor; (2) solidifying the hydrogel matrix material; (3) Performing cell culture to obtain the bone organoid.

4. The construction method according to claim 3, wherein: In step (1), the three-dimensional culture reactor is a three-dimensional culture reactor based on dynamic tensile stress.

5. The construction method according to claim 4, characterized in that: The three-dimensional culture reactor based on dynamic tensile stress is subjected to plasma spraying treatment to remove impurities on the inner surface and polylysine coating on the inner surface to improve the hydrophilicity and adhesion of the inner surface before adding the cell mixture.

6. The construction method according to claim 4, wherein: In step (2), the curing is light curing, the wavelength of light curing is 400-420nm, and the light curing time is 30-60s; In step (3), when cell culture is performed, the frequency of dynamic tensile stress is 0.5-0.8 Hz, the time is 2-3 h / day, and the ratio is 8-10%.

7. Use of the bone organoid according to claim 1 or 2 for preparing bone repair materials or in vitro research models.

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