Preparation method for composite bone matrix gelatin scaffold, and use of same

Abstract

Provided are a preparation method for a composite bone matrix gelatin scaffold and the use of same, the method comprising the following steps: step 1, mixing a synthetic peptide gel powder with sterile water to prepare an aqueous solution containing 15%-40% of a synthetic peptide substance, and metering the pH value thereof; step 2, placing a bone matrix gelatin scaffold prepared from mammalian ex-vivo bones into the aqueous solution obtained in step 1, so as to fully soak same; step 3, adding a sodium hydroxide solution to the aqueous solution of step 2 to regulate the pH value of the aqueous solution to be within the range of 7-8, and stopping adding the sodium hydroxide solution; step 4, placing into an incubator the aqueous solution obtained in step 3, in which the bone matrix gelatin scaffold is soaked and of which the pH value is in the range of 7-8, and observing the aqueous solution until the aqueous solution turns into a gel state; and step 5, taking out from said incubator the bone matrix gelatin scaffold of which both the surface and pores are covered by / filled with the synthetic peptide gel, and performing freeze drying to obtain the composite bone matrix gelatin scaffold.

Description

Preparation method of composite bone matrix gelatin scaffold and application thereof TECHNICAL FIELD

[0001] The present application relates to the field of biological medicine, and particularly relates to a preparation method of a composite bone matrix gelatin scaffold and application thereof. BACKGROUND

[0002] Human organoids are miniaturized and simplified versions of human organs cultured in vitro. 3D cell culture systems formed by pluripotent stem cells or tissue-derived progenitor cells through cell differentiation and self-organization can maximize the simulation of the tissue structure, cell type and specific function of real organs in vitro. This "minimal organ system" has great application potential in the fields of basic medical research of organ development, homeostasis, as well as disease modeling, drug screening and personalized medicine.

[0003] In the process of constructing cartilage organoids, a suitable scaffold carrier is selected to mimic the biological structure and growth environment of natural cartilage tissue, so as to generate stable cartilage organoids, i.e. organoids consistent with the physiological tissue structure of cartilage, cell differentiation type and function of secreting cartilage matrix, which are applied to basic medical research of cartilage differentiation and development and cartilage injury. The tissue engineering cartilage technology similar to the present technology is inconsistent with the actual problems solved and the purposes achieved by the present technology. The tissue engineering cartilage technology is to seed cartilage cells on a degradable scaffold material, and then implant the scaffold material into a cartilage defect site, so that the seeded cells form new cartilage in vivo to repair the defect site. The tissue engineering cartilage only requires that the seeded cartilage cells can proliferate well on the scaffold and maintain functions, and does not require that the cartilage morphology structure consistent with the physiological tissue structure is generated in vitro. TECHNICAL PROBLEM

[0004] The existing culture matrix for constructing cartilage organoids mostly uses natural or synthetic extracellular matrix gel products (collectively referred to as matrix glue) to support cell growth, such as using matrix glue Micromass for 3D culture of chondrocytes, but the results can only see different proliferation and differentiation of seed cells accumulated in the matrix glue microspheres, and no complete three-dimensional morphological structure of cartilage tissue is formed. The reason is that the matrix glue does not have a good three-dimensional porous structure and does not have a natural scaffold microenvironment for cartilage growth, which cannot generate a three-dimensional tissue structure consistent with the physiological structure. In the patent documents with publication numbers CN115645619A, CN1546654A, CN106075580A, etc., a natural bone matrix gelatin scaffold (English abbreviation BMG) is selected to prepare cartilage organoids from mammalian isolated bone. Although the bone matrix gelatin has a good three-dimensional porous structure and pore interconnection, the pore size range of 100-800 μm is too large for 10 μm sized seed cells, resulting in low adhesion of seed cells in the scaffold and easy loss, which cannot well meet the basic conditions for stable growth of cartilage organoids.

[0005] Tumor organoids are also a new type of preclinical tumor model system that can better reproduce the in vivo characteristics and heterogeneity of primary tumors, and are currently the most reliable experimental model for tumor disease modeling. Bone tumor and cartilage tumor organoids also face the problem of selecting appropriate biological scaffold carriers during construction. In addition to the above problems of lack of three-dimensional porous structure and too large pore size of the scaffold carrier in the culture process of cartilage organoids, the most prominent problem is to solve the problem of multiple growth factor sources. In the existing construction process of bone tumor and cartilage tumor organoids, in order to simulate the pathological microenvironment of in vivo high proliferation activity tumor tissue, different types and compositions of exogenous growth factors, chemical small molecule inhibitors / activators, etc. are added, making the culture system complex and expensive.

[0006] Due to the above reasons, the culture method of cartilage organoids, bone tumor and cartilage tumor organoids is not mature, the success rate is low, and the cost is high. So far, there are still few technical means that can stably construct cartilage organoids with structure and biological function simulation, and bone tumor and cartilage tumor organoids that can reproduce the pathological characteristics of patient tumor tissue. Technical solution

[0007] In order to overcome the shortcomings of the above prior art, the present application provides a composite bone matrix gelatin scaffold preparation method and its application in constructing cartilage organoids, bone tumor and cartilage tumor organoids, which can solve the problems of the existing scaffold materials not having a good three-dimensional structure with pore size and porosity, the pore size being too large resulting in low adhesion of seed cells, the addition of different types and compositions of exogenous growth factors resulting in a complex system and high cost, and the culture process being inaccurate, etc. which cannot stably construct the above organoids.

[0008] The application discloses a preparation method of a synthetic peptide gel composite bone matrix gelatin support material.

[0009] The synthetic peptide gel composite bone matrix gelatin support material:

[0010] Step 1, the synthetic peptide gel powder is configured into an aqueous solution with a synthetic peptide content of 15%-40% by adding sterile water;

[0011] Step 2, the aqueous solution is put into a vortex oscillator to be vortexed and oscillated, mixed uniformly to become a mixed aqueous solution, and the pH value is detected;

[0012] Step 3, the bone matrix gelatin support prepared from the mammal ex vivo bone is put into the mixed aqueous solution to be completely soaked;

[0013] Step 4, sodium hydroxide solution is gradually added in a small amount in the mixed aqueous solution in step 3, the pH value of the mixed aqueous solution is adjusted on the basis of the measured pH value, the pH value is continuously measured until the sodium hydroxide solution is stopped to be added when the pH value is in the range of 7-8;

[0014] Step 5, the mixed aqueous solution with the bone matrix gelatin support soaked in step 4 and with the pH value in the range of 7-8 is put into a 37 DEG C incubator, and the mixed aqueous solution is observed for 1-2 hours until the mixed aqueous solution becomes a gel state;

[0015] Step 6, the bone matrix gelatin support with the surface and the pores covered and filled with the synthetic peptide gel is taken out from the 37 DEG C incubator and is put into a 4 DEG C vacuum freeze dryer for 24-36 hours, and the composite bone matrix gelatin support is obtained after freeze drying.

[0016] Preferably, the synthetic peptide gel is an aldehyde polyaspartic acid (molecular formula abbreviation: CHO-PASP) gel, a methacrylamide gel, a silk fibroin or the like, and the aldehyde polyaspartic acid (CHO-PASP) gel is preferred.

[0017] Preferably, the mammal ex vivo bone in step 3 is selected from commonly used experimental animals such as mouse, rat, experimental rabbit, experimental small pig, experimental dog, experimental monkey and the like, and the experimental small pig ex vivo bone is preferred in view of applicability and cost.

[0018] Preferably, the bone matrix gelatin support prepared from the mammal ex vivo bone in step 3 has a three-dimensional porous structure and is rich in various growth factors for promoting proliferation and differentiation such as BMP, TGF-beta 1, IGF, FGF and the like, and the pore aperture is in the range of 100-700 μm.

[0019] The synthetic peptide gel composite bone matrix gelatin scaffold maintains the three-dimensional porous structure of the original bone matrix gelatin scaffold, is beneficial to the exchange of seed cell metabolites, is rich in various growth factors BMP, TGF-β1, IGF, FGF and the like which promote the proliferation and differentiation of seed cells, and reduces the pore size of the original bone matrix gelatin from 100-700 mu m to 10-20 mu m, thereby providing a stable growth condition and a space environment for the 10 mu m seed cells, improving the adhesion rate of the seed cells, and reasonably distributing the seed cells to avoid loss. Thus, the core problem of being unable to stably construct a cartilage organ, a bone tumor and a cartilage tumor organ is solved.

[0020] The application further discloses an application method of constructing a cartilage organ based on the composite bone matrix gelatin scaffold.

[0021] Step 1, sterilizing and disinfecting the prepared composite bone matrix gelatin scaffold and reserving it for later use;

[0022] Step 2, resuspending the cartilage seed cells to be inoculated in complete culture solution to obtain a cartilage cell suspension;

[0023] Step 3, preparing a cartilage organ culture container and an elastic soft gasket. The shape and size of the culture container can be determined according to the use of the generated cartilage organ; the elastic soft gasket has upper and lower surfaces and a peripheral surface with a certain height, and the shape, radial size of the upper and lower surfaces and the height should be suitable for the shape, size and diameter of the cartilage organ culture container. The soft gasket can be placed in the cartilage organ culture container, and the upper and lower surfaces of the elastic soft gasket are slightly larger than the size of the container diameter, and the elastic soft gasket is tightly sealed with the inner wall of the container by the elasticity of the elastic soft gasket, and is sealed in the required position of the cartilage organ culture container; a through hole is made at the center point of the elastic soft gasket according to the shape of the composite bone matrix gelatin scaffold, and the size of the through hole is slightly smaller than the size of the composite bone matrix gelatin scaffold; the cartilage organ culture container and the elastic soft gasket are sterilized and disinfected and reserved for later use;

[0024] Step 4, pressing the composite bone matrix gelatin scaffold reserved in step 1 into the center through hole of the elastic soft gasket, so that the upper surface of the composite bone matrix gelatin scaffold is flush with the upper surface of the elastic soft gasket, and the through hole periphery is tightly sealed with the composite bone matrix gelatin scaffold periphery by the elasticity; then the elastic soft gasket is sealed in the required position of the cartilage organ culture container as described in step 3; in this way, the liquid covering the composite bone matrix gelatin scaffold cannot leak along the inner wall of the culture container, nor can it seep out from the center through hole and the composite bone matrix gelatin scaffold sealing position;

[0025] Step 5, slowly adding the cartilage seed cell suspension to the composite bone matrix gelatin scaffold material in step 4, so that the composite bone matrix gelatin scaffold is fully infiltrated;

[0026] Step 6, the complete culture solution is added dropwise into the cartilage organ culture container to fully cover the composite bone matrix gelatin support, and the culture is carried out for 3-4 weeks to obtain the cartilage organ.

[0027] The biological characteristics of the selected cartilage seed cells in the present application are adherent cells, which grow by attaching or adhering to the surface of the support. Therefore, the composite bone matrix gelatin support is sealed in the container to avoid the seed cells added dropwise on the composite bone matrix gelatin support from leaking into the container below the elastic soft gasket and the composite bone matrix gelatin support, so as to ensure that the seed cells are fully infiltrated into the bone matrix gelatin support and improve the adhesion rate of the seed cells. This is one of the key culture links for stably constructing the cartilage organ and similar organoids in the present application, which is summarized from repeated experiments. We have tried to block the gap between the composite bone matrix gelatin support and the cartilage organ culture container with agarose gel and other biological gels, but the agarose gel and other biological gels will penetrate into the composite bone matrix gelatin support and affect the entry and adhesion of the seed cells, so it is impossible to stably construct the cartilage organ. We have also tried to use a centrifuge tube with a conical lower part, and cut the composite bone matrix gelatin support to a suitable size and use the slight expansion of the composite bone matrix gelatin support after absorbing the liquid to block it in the conical opening of the lower part of the centrifuge tube. However, due to the poor precision of manual cutting, the elasticity of the bone matrix gelatin after absorbing the liquid is too small, although it can be blocked, it cannot achieve the sealing of the gap, and a part of the seed cells still leaks into the container below along the gap between the composite bone matrix gelatin support and the inner wall of the cartilage organ culture container, which affects the growth success rate of the organoids.

[0028] Preferably, the composite bone matrix gelatin support in step 1 can be cut into various shapes such as cylindrical, rectangular, cubic and other regular and irregular shapes according to the needs of constructing the cartilage organ, and the cylindrical shape is preferred.

[0029] Preferably, the cartilage organ culture container in step 3 can be a cylinder, a cone, a cylinder-cone hybrid, etc., and the cylinder is preferred.

[0030] Preferably, the sealing mode of the cartilage organ culture container, the elastic soft gasket and the composite bone matrix gelatin support in steps 3 and 4 can refer to the several forms in FIG. 12, so that the seed cells are fully infiltrated into the composite bone matrix gelatin support under the action of gravity.

[0031] Preferably, in order to improve the efficiency and infiltration rate of the seed cells fully infiltrating into the composite bone matrix gelatin support material, the matching mode of the cartilage organ culture container and the elastic soft gasket, the composite bone matrix gelatin in steps 3 and 4 can also generate negative pressure by the device in FIG. 13 to compress and absorb the seed solution for full infiltration.

[0032] Preferably, the elastic soft seal pad in step 3 can be cylindrical, rectangular and other shapes that match the shape of the cartilage organ culture container, preferably cylindrical.

[0033] Preferably, the elastic soft seal pad in step 3 can be made of transparent or opaque materials, such as PVC soft rubber, silicone, polyurethane, etc., preferably transparent PVC soft rubber.

[0034] Preferably, the cartilage seed cells to be inoculated in step 2 are selected from primary cartilage cells or cartilage cell lines of human or animal origin in the exponential growth phase. The closest technology to the present application at present is the construction of tissue engineered cartilage for cartilage defect repair, but it uses bone marrow mesenchymal stem cells, adipose stem cells, etc. for cell inoculation, and needs to add various induction liquids to induce stem cells to differentiate into chondrocytes. The present application can preferably use primary cartilage cells or cartilage cell lines of human or animal origin in the exponential growth phase as seed cells, which are easy to obtain and do not require the addition of extra induction liquid to construct cartilage organs in vitro. In addition, the selection of seed cells in the exponential growth phase can ensure good seed cell viability and promote the stable construction of cartilage organs.

[0035] Preferably, the inoculation amount of the cartilage seed cells to be inoculated in step 2 is 7x10 6 -10x10 6 preferably per unit square millimeter of composite bone matrix gelatin scaffold. The present application found in multiple experiments that the inoculation density of seed cells is another key culture step that affects the stable construction of cartilage organs, and the seed cells should maintain an appropriate inoculation amount to obtain the best culture conditions. In the early stage, we used 1x10 6 -6x10 6 ATDC5 chondrocytes per 5mm (diameter) x 3mm (thickness) scaffold, and after culture, only undifferentiated ATDC5 chondrocytes were seen accumulated in the scaffold pore diameter, and no cartilage tissue with physiological structure was formed (see Figure 5). This is because the low density of seed cells cannot proliferate and differentiate rapidly within a certain period of time, and there is a lack of interaction between cells, leading to slow cell proliferation and differentiation. In addition, we used 11x10 6 -12x10 6 ATDC5 chondrocytes per 5mm (diameter) x 3mm (thickness) scaffold, and found that a large number of inoculated chondrocytes accumulated above the scaffold, and no cartilage organ tissue was formed after culture (see Figure 5). This is because too many seed cells can easily lead to cell aggregation to form cell aggregates, making it difficult for cells to enter the inside of the scaffold and attach to the scaffold; on the other hand, under high density conditions, the nutrients and space for seed cells become limited, leading to slow growth or even death of cells, thereby hindering the construction of cartilage organs.

[0036] Preferably, after inoculating the cartilage seed cells in step 6, the culture is carried out for 3-4 weeks, preferably 4 weeks. In many experiments, it is found that after 1 week of culture, only the seed cells are attached to the bone matrix gel holes; after 2 weeks of culture, the seed cells in the bone matrix gel holes proliferate significantly, but no tissue structure is formed; after 3 weeks of culture, the cartilage organoids consistent with the physiological structure are obtained; and after 4 weeks of culture, the obtained cartilage organoids have more complete structure, the cells are arranged in order, and the cartilage organoids have stable tissue structure and biological function (Figure 6). No obvious change in the structure of the new cartilage organoids is observed after 5 weeks of culture.

[0037] In the embodiments of the present application, the inoculation of cartilage seed cells is preferably carried out for 4 weeks, and the generated cartilage organoids have a diameter of 2-5 mm (Figure 4), which is much larger than the size of the cartilage organoids generated by the prior art, and thus is more consistent with the human physiological structure and state, and has better beneficial effects for subsequent research on cartilage injury and differentiation. The prior art relies on extracellular matrix gel products to prepare microspheres as culture substrates to construct organoids. Due to the small size of the microspheres, the organoids constructed by the microspheres often have a diameter of only 100-500 μm.

[0038] The present application also discloses an application method for constructing bone tumor and cartilage tumor organoids based on the above-mentioned composite bone matrix gel scaffold, comprising the following steps:

[0039] Step 1: The composite bone matrix gel scaffold is sterilized and prepared for standby use;

[0040] Step 2: The primary bone / cartilage sarcoma cells or bone / cartilage sarcoma cell lines to be inoculated are resuspended in complete culture medium to obtain a cell suspension;

[0041] Step 3: A bone tumor or cartilage tumor organoid culture container and a flexible soft gasket are prepared. The shape and size of the culture container can be determined according to the purpose of generating bone tumor or cartilage tumor organoids; the flexible soft gasket has upper and lower surfaces and a peripheral surface with a certain height, and the shape, size of the upper and lower surfaces and the height should be suitable for the shape, size of the bone tumor or cartilage tumor organoid culture container. The soft gasket can be placed in the bone tumor or cartilage tumor organoid culture container, and the upper and lower surfaces of the soft gasket are slightly larger than the size of the container, and the flexible soft gasket is tightly sealed with the inner wall of the container by the elasticity of the flexible soft gasket, and is placed in the required position of the bone tumor or cartilage tumor organoid culture container; a through hole is made at the center point of the flexible soft gasket according to the shape of the composite bone matrix gel, and the size of the through hole is slightly smaller than the size of the composite bone matrix gel. The bone tumor or cartilage tumor organoid culture container and the flexible soft gasket are sterilized and prepared for standby use;

[0042] Step 4, press the prepared composite bone matrix gelatin scaffold in step 1 into the center hole of the elastic soft gasket, so that the upper surface of the composite bone matrix gelatin scaffold material is flush with the upper surface of the elastic soft gasket, and the elasticity of the center hole makes the periphery of the center hole tightly sealed with the periphery of the composite bone matrix gelatin, and then place the elastic soft gasket in the desired position of the bone tumor or cartilage tumor organoid culture container according to step 3, so that the liquid covering the composite bone matrix gelatin scaffold material cannot leak along the inner wall of the culture container, nor can it seep out from the sealing part of the center hole and the composite bone matrix gelatin;

[0043] Step 5, drop the bone tumor or cartilage tumor seed cell suspension onto the composite bone matrix gelatin scaffold in step 4, so that it completely infiltrates the composite bone matrix gelatin scaffold;

[0044] Step 6, further drop the complete culture medium into the bone tumor or cartilage tumor organoid culture container, fully covering the bone matrix gelatin scaffold, and culture for 1-4 weeks to obtain a cartilage organoid.

[0045] Preferably, the bone matrix gelatin in the composite bone matrix gelatin scaffold material in step 1 is selected from a scaffold material derived from mammalian in vitro cancellous bone rich in various growth factors such as BMP, TGF-β1, IGF, FGF, etc. that promote proliferation and differentiation.

[0046] Preferably, the bone / cartilage tumor cells in step 2 are derived from a mammal.

[0047] Preferably, the primary bone / cartilage tumor cells in step 2 are derived from the diseased side of the bone / cartilage tumor subject and are cultured in vitro.

[0048] Preferably, the primary bone / cartilage tumor cells in step 2 are derived from the diseased side of the bone / cartilage tumor disease model animal and are cultured in vitro.

[0049] Preferably, the bone tumor cell lines in step 2 include human osteosarcoma cell lines 143b, MG-63, Saos-2, U2OS, and mouse osteosarcoma osteoblast cell line K7M2-WT; and the cartilage tumor cell lines include human chondrosarcoma cell lines SW1353, HCS-2 / 8, CAL-78, and mouse chondrosarcoma cell line EHS.

[0050] Preferably, the bone / cartilage tumor organoid culture time in step 6 is preferably 1-3 weeks, and most preferably 2 weeks. Beneficial effects

[0051] Compared with the prior art, the present application has the following beneficial effects:

[0052] First, the application of the key link technology method in the preparation of the composite bone matrix gelatin solves the technical problems that the prior art cannot stably construct cartilage organoids, bone tumors and cartilage tumor organoids. The cartilage organoids (see Figure 3) we construct have typical normal physiological structures, and the articular cartilage-like cartilage and the epiphyseal plate-like cartilage have typical normal physiological structures. The articular cartilage-like cartilage can be seen to have surface, middle and deep cartilage morphological structures, and the epiphyseal plate-like cartilage can be seen to have resting layer, proliferation layer, hypertrophy layer and calcification layer, and even new bone trabeculae, which have complete tissue structure and stable biological function, and can simulate the physiological structure and function of human cartilage to the greatest extent. At the same time, the cell diameter of the cartilage organoids constructed by the application can reach 2-5 mm (Figure 4), which is much larger than the 100-500 μm of the organoids constructed by the prior art, and is more in line with the physiological structure and state of the human body, and the accuracy and precision are better when applied to cartilage injury and differentiation research and organ development, homeostasis and regeneration basic medical research. The bone tumor and cartilage tumor organoids we construct have high consistency with the original tumor tissue characteristics (Figures 8, 9 and 10), and the cell diameter of the organoids can reach about 2 mm (see Figure 14), which is highly consistent with the human-derived tissue structure, molecular characteristics, protein expression pattern and drug response, has similar genomic, transcriptomic, morphological and functional characteristics with patient tumor tissue, and can highly reproduce the original characteristics of the tumor in the body in vitro, thereby providing an effective model for drug sensitivity research of tumor patients and precise targeted drug treatment.

[0053] Second, the composite bone matrix gelatin scaffold prepared by the application not only retains the three-dimensional porous structure of bone matrix gelatin and various growth factors such as BMP, TGF-β1, IGF, FGF that promote the proliferation and differentiation of organoids, but also has excellent in vitro induction ability to form organoids, does not need to add additional growth factors, and can stably construct organoids using conventional complete culture solution of corresponding seed cells, which is simple and easy to operate and low in cost. In addition, the pore size is reduced to the range of 10-20 μm, which meets the basic growth requirements of seed cells with a size of about 10 μm, and overcomes the defects that the excessively large pore size of 100-700 μm causes the seed cells to be difficult to adhere and easily lost, thereby failing to stably construct organoids.

[0054] Third, the application solves the problems of insufficient infiltration of seed cells into the composite bone matrix gelatin scaffold, improper seed cell inoculation concentration and improper culture time, accurately grasps the key culture link, forms a mature culture process, and greatly improves the success rate of constructing cartilage organoids and bone / cartilage tumor organoids. BRIEF DESCRIPTION OF DRAWINGS

[0055] Figure 1 is a scanning electron microscope image of the pore size of the aldehyde polyaspartic acid (CHO-PASP) gel composite bone matrix gelatin scaffold;

[0056] Figure 2 is a comparison chart of cartilage organoids generated with bone matrix gelatin scaffolds and cartilage organoids generated with bone matrix gelatin scaffolds complexed with CHO-PASP gel;

[0057] Figure 3 is a staining chart of articular cartilage organoids and epiphyseal cartilage organoids;

[0058] Figure 4 is a chart of the diameter range of constructed articular cartilage organoids;

[0059] Figure 5 is a chart of the effect of cell seeding density on the formation of cartilage organoids;

[0060] Figure 6 is a chart of the effect of different culture times on the formation of cartilage organoids;

[0061] Figure 7 is a chart of bone tumor organoids;

[0062] Figure 8 is a chart of the histomorphological structure of bone tumor organoids;

[0063] Figure 9 is a chart of the staining identification of bone tumor organoids;

[0064] Figure 10 is a chart of the histomorphological structure of cartilage tumor organoids;

[0065] Figure 11 is a chart of the use method of cartilage / bone tumor / cartilage tumor organoid culture vessels;

[0066] Figure 12 is a chart of the use method of cartilage / bone tumor / cartilage tumor organoid culture vessels;

[0067] Figure 13 is a chart of the use method of cartilage / bone tumor / cartilage tumor organoid culture vessels;

[0068] Figure 14 is a chart of the diameter range of bone tumor organoids;

[0069] Figure 15 is a chart of the molecular structure of CHO-PASP;

[0070] Reference signs: 1 - composite bone matrix gelatin scaffold; 2 - elastic soft gasket; 3 - culture vessel.

[0071] Enter the best mode of the invention here. Embodiment of the present invention

[0072] The present invention will be further described in detail below in conjunction with the accompanying drawings 1-15.

[0073] In order to enable those skilled in the art to better understand the present invention, the technical solutions in the embodiments of the present invention will be described clearly and completely below in conjunction with the accompanying drawings in the embodiments of the present invention:

[0074] Example 1: Preparation method of composite bone matrix gelatin scaffold

[0075] Preparation method of synthetic peptide gel composite bone matrix gelatin scaffold

[0076] 0.3 g of sterile aldehyde polyaspartic acid (CHO-PASP) gel powder was weighed into a 5 mL sterile centrifuge tube, and then 0.7 mL of sterile water was added.

[0077] The mixture was observed to be uniformly mixed into an aldehyde polyaspartic acid (CHO-PASP) gel aqueous solution using a vortex shaker. Then, pH test paper was used to detect the pH value of the aldehyde polyaspartic acid (CHO-PASP) gel aqueous solution, which was about 4-5.

[0078] Next, a sterile bone matrix gelatin scaffold with a diameter of 5 mm and a thickness of 3 mm prepared from the experimental minipig's isolated cancellous bone was placed in the aldehyde polyaspartic acid (CHO-PASP) gel aqueous solution described above, and after complete immersion, a small amount of 1 M sodium hydroxide solution was gradually added to the aldehyde polyaspartic acid (CHO-PASP) gel aqueous solution containing the bone matrix gelatin scaffold, and the pH value of the aldehyde polyaspartic acid (CHO-PASP) gel aqueous solution was continuously detected during the process until the pH value was adjusted to 7.4, at which point the addition of sodium hydroxide solution was stopped. Then, the aldehyde polyaspartic acid (CHO-PASP) gel aqueous solution containing the bone matrix gelatin scaffold with the adjusted pH value was placed in a 37°C incubator for observation, and after about 1 hour, it was observed that the aqueous solution had become a gel state.

[0079] At this time, the aldehyde polyaspartic acid (CHO-PASP) gel had covered the surface of the bone matrix gelatin and filled the pores in the bone matrix gelatin scaffold, and the compound of the bone matrix gelatin was removed with tweezers and placed in a 4°C vacuum freeze dryer for 24 hours of freeze-drying, thereby obtaining a composite bone matrix gelatin scaffold.

[0080] 2) Structural characteristics of the composite bone matrix gelatin scaffold

[0081] The freeze-dried composite bone matrix gelatin scaffold was fixed by gold spraying, and the structural characteristics and pore size of the composite bone matrix gelatin scaffold were scanned using a scanning electron microscope. The results showed that the composite bone matrix gelatin (BMG) scaffold filled with aldehyde polyaspartic acid (CHO-PASP) gel presented a three-dimensional porous structure with interconnected channels, as shown in Figure 1: the left image shows the structure of the composite bone matrix gelatin under an electron microscope, and the right image shows the size distribution of the pores of the composite bone matrix gelatin scaffold, most of which have a size of about 10-20 μm.

[0082] The measured nutritional factors of the composite bone matrix gelatin scaffold are shown in Table 1.

[0083] Example 2: Preparation of joint cartilage organoids using a composite bone matrix gelatin scaffold

[0084] 1) A method for constructing an articular cartilage organoid using composite bone matrix gelatin scaffolds, comprising the following steps:

[0085] (1) Select a composite bone matrix gelatin scaffold 1 and cut it into a cylinder with a diameter of 5 mm and a thickness of 3 mm. First, irradiate it with ultraviolet light for 48 hours (24 hours on each side) in a clean bench, and then soak it in PBS containing 100 U / mL penicillin and 100 μg / mL streptomycin for sterilization and standby;

[0086] (2) Take the C28 / I2 chondrocytes in the exponential growth phase, perform routine digestion with trypsin, and collect the cells. Resuspend the cells in complete culture medium (DMEM / F12 culture medium containing 5% fetal bovine serum, 100 U / mL penicillin, and 100 μg / mL streptomycin) and adjust the concentration to 8×10 6

[0087] (3) Prepare a 5 mL cylindrical round-bottom glass centrifuge tube 3 with a diameter of 8.5 mm and a height of 41 mm, and a cylindrical elastic transparent silicone rubber gasket 2 with a diameter of 8.55 mm on the top and bottom and a thickness of 4 mm. Drill a cylindrical hole with a diameter of 4.9 mm in the center of the gasket 2.5 mm in radius. Sterilize and disinfect the cylindrical round-bottom centrifuge tube 3 and the cylindrical elastic gasket 2 for standby;

[0088] (4) Take the standby cylindrical composite bone matrix gelatin scaffold 1 with a diameter of 5 mm and a thickness of 3 mm in (1), discard the PBS covering it, take it out and press it into the center hole of the elastic gasket 2, so that the upper surface of the composite bone matrix gelatin 1 is flush with the upper surface of the gasket. The composite bone matrix gelatin scaffold 1 with a diameter of 5 mm and a thickness of 3 mm is tightly sealed in the soft gasket hole with a diameter of 4.9 mm and a thickness of 4 mm due to its elasticity. Then, press the cylindrical elastic gasket 2 with the composite bone matrix gelatin scaffold 1 tightly sealed in it uniformly to a distance of 3 mm from the bottom of the 5 mL cylindrical round-bottom centrifuge tube 3 with a diameter of 8.5 mm and a height of 41 mm. The elastic gasket 2 with a diameter of 8.55 mm is tightly sealed in the centrifuge tube 3 with a diameter of 8.5 mm due to its elasticity. As shown in FIG. 11

[0089] (5) Slowly drop 1 mL of the prepared C28 / I2 chondrocyte suspension vertically onto the composite bone matrix gelatin scaffold 1 at the bottom of the centrifuge tube, and let it stand for 10 minutes. Observe whether it is fully soaked in the composite bone matrix scaffold material 1;

[0090] ​(6) Then, 3 mL of complete culture solution was added dropwise into the cylindrical round-bottom centrifuge tube 3 to fully cover the composite bone matrix gelatin scaffold 1, which was placed in the incubator for culture. The complete culture solution was replaced every two days, and the culture was performed for 4 weeks.

[0091] (7) After the culture was completed, the culture solution was discarded, and the elastic soft gasket 2 was pulled out of the cylindrical round-bottom centrifuge tube 3 uniformly and forcibly. The tissue block in the central through hole of the elastic soft gasket 2 was taken out, and the articular cartilage organoid was obtained.

[0092] Fig. 2 is a left side view of a cartilage organoid generated by using a bone matrix gelatin scaffold, and a right side view of a cartilage organoid generated by using a composite bone matrix gelatin scaffold 1. The right side view shows that the cartilage organoid on the right side has a more compact arrangement of chondrocytes, more obvious chondral lacunae, and more abundant extracellular matrix, and has the typical characteristics of scattered arrangement of chondrocytes.

[0093] 2) Identify the morphological structure of the articular cartilage organoid tissue.

[0094] The articular cartilage organoid obtained by the culture in Example 2 was fixed with 4% paraformaldehyde for 30 minutes, and then was preserved by being embedded in a wax block after being dehydrated by a conventional alcohol gradient.

[0095] A 5-μm paraffin section was prepared, and after being deparaffinated and dehydrated, the section was subjected to conventional H&E staining and TB staining, and then was placed under an optical microscope to take photographs and be observed, as shown in Fig. 3A. The results show that the newly generated surface chondrocytes of the constructed articular cartilage organoid are flat, the middle layer chondrocytes are oval or round, and the deep layer chondrocytes are hypertrophic. The chondrocytes in each layer are arranged in an orderly and regular manner, forming a surface layer, a middle layer and a deep layer structure consistent with the physiological articular cartilage, and have obvious differentiation period and zone characteristics. The TB staining results show that the extracellular matrix of the constructed articular cartilage organoid is blue-violet and uniformly distributed, indicating that the constructed articular cartilage organoid has the normal cartilage matrix secretion characteristics. Figs. 4a and 4b show that the diameter of the constructed articular cartilage organoid is about 2-5 mm.

[0096] Example 3: Application of the composite bone matrix gelatin scaffold 1 to construct an epiphyseal plate cartilage organoid

[0097] 1) The method for constructing an epiphyseal plate cartilage organoid by using the composite bone matrix gelatin scaffold 1 comprises the following steps:

[0098] (1) The composite bone matrix gelatin scaffold 1 was cut into a cylindrical body with a diameter of 5 mm and a thickness of 3 mm, and was first subjected to ultraviolet irradiation for 48 hours (24 hours for each side) in an ultraclean workbench, and then was sterilized by being immersed in PBS containing 100 U / mL penicillin and 100 μg / mL streptomycin, and was reserved for use.

[0099] (2) Take the ATDC5 chondrocyte in exponential growth phase, digest it with trypsin, and collect the cells. Resuspend the cells in complete culture medium (DMEM / F12 culture medium containing 10% fetal bovine serum, 100 U / mL penicillin, and 100 μg / mL streptomycin) to adjust the concentration to 9 x 10 6

[0100] (3) Prepare a 5 mL cylindrical round-bottom glass centrifuge tube 3 with a diameter of 8.5 mm and a height of 41 mm, and a cylindrical elastic transparent silicone rubber gasket 2 with a diameter of 8.55 mm and a thickness of 4 mm. Drill a cylindrical through hole with a diameter of 4.9 mm at a radius of 2.45 mm in the center of the gasket. Sterilize the cylindrical round-bottom centrifuge tube 3 and the cylindrical elastic gasket 2, and reserve them for later use;

[0101] (4) Discard the PBS covering the diameter 5 mm, thickness 3 mm composite bone matrix gelatin scaffold 1 material reserved in (1), take it out, and press it into the central through hole of the elastic gasket 2 so that the upper surface of the composite bone matrix gelatin scaffold 1 is flush with the upper surface of the gasket. The diameter 5 mm, thickness 3 mm composite bone matrix gelatin scaffold 1 is tightly sealed in the diameter 4.9 mm, thickness 4 mm through hole of the gasket due to elasticity. Then, press the cylindrical elastic gasket 2 with the sealed composite bone matrix gelatin scaffold 1 into the 5 mL cylindrical round-bottom centrifuge tube 3 with a diameter of 8.5 mm and a height of 41 mm at a distance of 3 mm from the bottom. The diameter 8.55 mm elastic gasket 2 is tightly sealed in the diameter 8.5 mm centrifuge tube due to elasticity, as shown in FIG. 11;

[0102] (5) Vertically and slowly drop 1 mL of the prepared ATDC5 chondrocyte suspension onto the composite bone matrix gelatin scaffold 1 at the bottom of the centrifuge tube, and stand for 10 minutes to observe whether it is fully soaked in the scaffold.

[0103] (6) Drop 3 mL of complete culture medium into the round-bottom centrifuge tube 3 to fully cover the composite bone matrix gelatin scaffold 1, and place it in an incubator. Replace the culture medium every two days, and culture for 4 weeks.

[0104] (7) After the culture is completed, discard the culture medium, and uniformly and forcefully pull out the elastic gasket 2 from the cylindrical round-bottom centrifuge tube 3. Take out the tissue block in the central through hole of the elastic gasket 2 to obtain the epiphyseal cartilage organoid tissue.

[0105] 2) Identify the morphological structure of the epiphyseal cartilage organoid tissue.

[0106] ​(1) The epiphyseal plate cartilage organoid tissue obtained by culture in Example 3 above was fixed with 4% paraformaldehyde for 30 minutes, and after routine alcohol dehydration, it was embedded in a wax block for preservation.

[0107] (2) 5-μm paraffin sections were prepared, and after routine dewaxing and dehydration, they were stained with routine H&E and TB, and then placed under an optical microscope for photographing and observation, as shown in FIG. 3B. The results showed that H&E staining showed that the epiphyseal plate cartilage organoid had obvious proliferation of new cartilage cells, which were oval or round. The cartilage cells were arranged in an orderly and regular manner, forming a columnar arrangement structure similar to the normal in-vivo epiphyseal plate cartilage, and had obvious differentiation stages and zone characteristics. The upper layer cells in the new tissue of the epiphyseal plate cartilage organoid were small in size and less in number, which were close to the in-vivo epiphyseal plate cartilage resting layer cartilage cells; the middle layer cells were active in proliferation, oval in shape, large in size, and arranged in a longitudinal columnar manner, which were close to the in-vivo epiphyseal plate cartilage proliferative layer cartilage cells; and the deep layer cells were large in size and round in shape, arranged in a longitudinal columnar manner, which were close to the in-vivo epiphyseal plate cartilage proliferative layer cartilage cells. TB staining showed that the extracellular matrix of the formed epiphyseal plate cartilage organoid was blue-purple and uniformly distributed, and the matrix around the deep layer of the hypertrophic cells was darkened, indicating that the epiphyseal plate cartilage had the secretion characteristics of cartilage tissue. FIGS. 4c and 4d show that the constructed epiphyseal plate cartilage organoid had a diameter of about 2 mm-5 mm, reaching the millimeter level.

[0108] Example 4: Preparation of bone tumor and cartilage tumor organoids using composite bone matrix gelatin scaffold 1.

[0109] 1. Preparation of osteosarcoma organoids using composite bone matrix gelatin scaffold 1.

[0110] 1) The composite bone matrix gelatin scaffold 1 was cut into a cylinder with a diameter of 5 mm and a thickness of 3 mm, and then subjected to ultraviolet irradiation for 48 hours (24 hours on each side) in a clean bench, and then sterilized by immersion in PBS containing 100 U / mL penicillin and 100 μg / mL streptomycin for standby;

[0111] 2) The logarithmic growth phase human osteosarcoma U2OS cell line was trypsinized and the cells were collected. The cells were resuspended in McCoy's 5A complete culture medium (containing 10% fetal bovine serum, 100 U / mL penicillin and 100 μg / mL streptomycin), and the concentration was adjusted to 8×10 6

[0112] ​3) Prepare a 5 mL cylindrical round-bottom glass centrifuge tube 3 with a diameter of 8.5 mm and a height of 41 mm, and a cylindrical elastic transparent silicone rubber gasket 2 with a diameter of 8.55 mm and a thickness of 4 mm. Drill a cylindrical hole with a diameter of 4.9 mm in the center of the gasket 2, and sterilize the cylindrical round-bottom centrifuge tube 3 and the cylindrical elastic gasket 2 for later use;

[0113] 4) Discard the PBS covering the diameter 5 mm, thickness 3 mm composite bone matrix gelatin scaffold 1 prepared in step 1, and press the scaffold into the center hole of the elastic gasket 2 so that the upper surface of the scaffold and the upper surface of the gasket are flush. The diameter 5 mm, thickness 3 mm composite bone matrix gelatin scaffold 1 is tightly sealed in the diameter 4.9 mm, thickness 4 mm hole of the gasket. Then, press the gasket with the scaffold tightly into the 5 mL cylindrical round-bottom centrifuge tube 3 at a distance of 3 mm from the bottom of the tube. The diameter 8.55 mm, thickness 4 mm gasket is tightly sealed in the diameter 8.5 mm centrifuge tube 3. See Fig. 11.

[0114] 5) Slowly add 1 mL of U2OS cell suspension vertically onto the scaffold material at the bottom of the centrifuge tube 3, and let it stand for 10 minutes. Observe whether the cells are fully immersed in the scaffold.

[0115] 6) Add 3 mL of McCoy's 5A complete medium, and culture in a 37°C, 5% CO2 incubator for 2 weeks. Replace the medium every day. After the culture is completed, discard the medium, and pull out the gasket 2 from the tube 3. Take out the tissue block in the center hole of the gasket 2, and obtain the osteosarcoma organoid. See Fig. 7.

[0116] 2. Identification of osteosarcoma organoid tissue morphological structure

[0117] 1) Fix the osteosarcoma organoid obtained in step 1 of Example 4 with 4% paraformaldehyde for 30 minutes, and perform conventional gradient dehydration and wax block embedding. Prepare 5 μm paraffin sections, and perform conventional dewaxing and hydration. Then, perform hematoxylin and eosin staining, and dehydrate and mount the sections after the staining.

[0118] Fix the osteosarcoma organoid obtained in step 1 of Example 4 with 4% paraformaldehyde for 30 minutes, and perform conventional gradient dehydration and wax block embedding. Prepare 5 μm paraffin sections, and perform conventional dewaxing and hydration. Then, perform H&E, Masson, Sirius red staining, and ALP, OCN immunohistochemical staining, and dehydrate and mount the sections after the staining.

[0119] 2) Microscopic observation under an optical microscope, as shown in Figure 8a. The microscopic examination reveals that osteosarcoma organoid tissue structures can be formed on and within the pores of the bone matrix gelatin 1 scaffold. The tumor cells are irregularly shaped, exhibiting significant atypia and pleomorphism, with abnormally enlarged nuclei and readily visible mitotic figures. Furthermore, a small amount of irregular mesh-like bone-like tissue and woven bone formation are present between the tumor cells. These tissue structures are consistent with the morphological structure of osteosarcoma tumors in patients (as shown in Figure 8b), indicating that osteosarcoma organoids have been successfully and stably constructed based on this invention.

[0120] The images were taken under an optical microscope, as shown in Figure 9. Masson staining stained new osteoblasts blue and mature osteoblasts blue (Figure 9a); Sirius red staining stained osteoblasts red, showing collagen fiber formation (Figure 9b); ALP and OCN immunohistochemical staining stained osteoblasts brown (Figure 9c).

[0121] All the above identifications indicate that the constructed organoid is an osteosarcoma organoid.

[0122] 3. Preparation of chondrosarcoma organoids using composite bone matrix gelatin scaffold 1

[0123] 1) Select composite bone matrix gelatin scaffold 1 and cut it into cylinders with a diameter of 5 mm and a thickness of 3 mm. First, irradiate it with ultraviolet light in a clean bench for 48 hours (24 hours for each side). Then, sterilize it by soaking it in PBS containing 100 U / mL penicillin and 100 μg / mL streptomycin for later use.

[0124] 2) Human chondrosarcoma SW1353 cells in logarithmic growth phase were harvested, routinely digested with trypsin, and the cells were collected. The cells were resuspended in SW1353 cell-specific medium (L15) (containing 10% fetal bovine serum, 100 U / mL penicillin, and 100 μg / mL streptomycin), and the concentration was adjusted to 9 × 10⁻⁶. 6 per mL.

[0125] 3) Prepare a 5 mL cylindrical glass centrifuge tube 3 with a diameter of 8.5 mm and a height of 41 mm, and a cylindrical flexible transparent silicone soft seal pad 2 with a top and bottom diameter of 8.55 mm and a thickness of 4 mm. Carve a cylindrical through-hole with a diameter of 4.9 mm around the center of the soft seal pad with a radius of 2.45 mm. Sterilize the cylindrical centrifuge tube 3 and the cylindrical flexible soft seal pad 2 before use.

[0126] 4) Take out the composite bone matrix gelatin scaffold 1 in 1) and press into the center hole of the elastic soft seal 2, so that the upper surface of the composite bone matrix gelatin scaffold 1 and the upper surface of the soft seal are flush, and the composite bone matrix gelatin scaffold 1 with a diameter of 5 mm and a thickness of 3 mm is tightly sealed in the hole of the soft seal with a diameter of 4.9 mm and a thickness of 4 mm; then press the soft seal 2 with a diameter of 8.55 mm and a thickness of 4 mm, which is sealed with the composite bone matrix gelatin scaffold 1, to the bottom of the 5 mL cylindrical round-bottom centrifuge tube 3 at a distance of 3 mm from the bottom, and the soft seal 2 with a diameter of 8.55 mm is tightly sealed in the centrifuge tube 3 with a diameter of 8.5 mm, as shown in FIG. 11;

[0127] 5) Slowly drop 1 mL of SW1353 cell suspension vertically onto the composite bone matrix gelatin scaffold 1 at the bottom of the centrifuge tube 3, and let it stand for 10 minutes to observe whether it is fully soaked in the scaffold;

[0128] 6) Add 4 mL of SW1353 cell culture medium (L15) and culture in a 37°C, 5% CO2 incubator for 2 weeks, replacing the fresh medium every day. After the culture is completed, discard the culture solution, and pull out the elastic soft seal 2 from the cylindrical round-bottom centrifuge tube 3, and take out the tissue block in the center hole of the elastic soft seal 2, to obtain the chondrosarcoma organoid. As shown in FIG. 10a.

[0129] 4. Identification of chondrosarcoma organoid tissue morphological structure

[0130] 1) The chondrosarcoma organoid obtained by culturing 3 in Example 4 above is fixed with 4% paraformaldehyde for 30 minutes, and then subjected to conventional gradient dehydration and wax block embedding. Prepare 5 μm paraffin sections, and after conventional dewaxing and hydration, perform hematoxylin and eosin staining, and after staining, dehydrate and mount.

[0131] 2) Take photos under an optical microscope, as shown in FIG. 10a, and the microscope shows that chondrosarcoma organoid-like tissue structures can be formed on the composite bone matrix gelatin scaffold 1 and within the scaffold aperture, the tumor cells are abundant, the cell nuclei are of different sizes, irregularly dark, and double nuclei, multinucleated, atypical nuclei and mitotic figures can be seen, the cartilage matrix is abundant, often accompanied by calcification and ossification. The chondrosarcoma organoid constructed is consistent with the morphological structure of chondrosarcoma patient tissue (as shown in FIG. 10b), indicating that the constructed organoid is a chondrosarcoma organoid.

[0132] The embodiment is only an explanation of the application, which is not a limitation of the application. Those skilled in the art can make modifications to the embodiment without creative contribution, as long as the modifications are within the protection scope of the application.

[0133] Table 1 Bone matrix gel 1 scaffold protein profile growth factor detection

[0134] Gene name Protein full name Protein size -10 lg PBMP1 Metalloendopeptidase 11 64 149.13 IGF2 Insulin-like growth factor II 20 31 133 8.72 IGFBP1 Insulin-like growth factor-binding protein 1 283 135.58 IGFBP3 Insulin-like growth factor-binding protein 3 316 906 6.63 IGFBP5 Insulin-like growth factor-binding protein 5 303 317 64.03 TGFB1 Transforming growth factor beta-1 44 280 42.63 TGFB2 Transforming growth factor beta-2 49 922 26.44 TGFB1 Transforming growth factor-beta-induced protein ig-h3 744 621 00.94 TGFB2 Transforming growth factor beta 50 661 126.44 FGFR1 Fibroblast growth factor receptor 9 160 638.63

Claims

1. A method of preparing a composite bone matrix gelatin scaffold, characterized by, The method comprises the following steps: Step 1, uniformly mix the synthetic peptide gel powder with sterile water to prepare a water solution with the synthetic peptide accounting for 15-40%, and detect the pH value of the water solution; Step 2, soak the bone matrix gel scaffold prepared from the mammal bone in the water solution obtained in step 1 until the water solution is fully soaked; Step 3, gradually add sodium hydroxide solution to the water solution in step 2 to adjust the pH value of the water solution until the pH value is in the range of 7-8, and stop adding the sodium hydroxide solution; Step 4, place the water solution obtained in step 3 in which the bone matrix gel scaffold is soaked and the pH value is in the range of 7-8 in a culture box, and observe the water solution until the water solution becomes a gel state; Step 5, take out the bone matrix gel scaffold in which the surface and pores are covered and filled with the synthetic peptide gel in the culture box in step 4, and freeze-dry the bone matrix gel scaffold to obtain a composite bone matrix gel scaffold (1).

2. The method of claim 1, wherein the composite bone matrix gelatin scaffold is prepared by the steps of: The synthetic peptide gel in step 1 comprises aldehyde polyaspartic acid gel or methacrylamide gel or silk fibroin.

3. The method of claim 1, wherein the composite bone matrix gelatin scaffold is prepared by the steps of: The mammal bone in step 2 is the bone of a mouse, a rat, a laboratory rabbit, a laboratory piglet, a laboratory dog or a laboratory monkey.

4. The method of claim 1, wherein the composite bone matrix gelatin scaffold is prepared by the steps of: The pore size of the bone matrix gel scaffold prepared from the mammal bone in step 2 is in the range of 100-700 μm.

5. The method of using the composite bone matrix gelatin scaffold to construct a cartilage organoid according to any one of claims 1-4, characterized in that, The method comprises the following steps: Step 1, sterilize and disinfect the composite bone matrix gel scaffold (1) prepared in any one of claims 1-4 for standby; Step 2, resuspend the cartilage seed cells to be inoculated in complete culture solution to obtain a cartilage cell suspension; Step 3, prepare a cartilage organ culture container (3) and an elastic soft gasket (2); the shape and size of the culture container can be determined according to the purpose of generating the cartilage organ; the elastic soft gasket (2) has upper and lower surfaces and a periphery with a certain height, and the shape, the size of the upper and lower surfaces and the height are adapted to the shape and size of the cartilage organ culture container (3); the elastic soft gasket (2) can be placed in the cartilage organ culture container (3), the radial size of the elastic soft gasket (2) is greater than the size of the culture container (3), the elastic soft gasket (2) is tightly sealed with the inner wall of the culture container (3) by using the elasticity of the elastic soft gasket (2), and is sealed at a required position in the cartilage organ culture container (3); a through hole is made at the center point of the elastic soft gasket (2) according to the shape of the composite bone matrix gel (1), and the size of the through hole is smaller than the size of the composite bone matrix gel (1); the cartilage organ culture container (3) and the elastic soft gasket (2) are sterilized and disinfected for standby; Step 4, press the composite bone matrix gel scaffold (1) for standby in step 1 into the center through hole of the elastic soft gasket (2), tightly seal the periphery of the center through hole with the periphery of the composite bone matrix gel (1) by using the elasticity of the center through hole, and then seal the elastic soft gasket (2) at the required position in the cartilage organ culture container (3) according to step 3, so that the liquid covering the composite bone matrix gel (1) cannot leak along the inner wall of the culture container (3) or seep out from the sealing position of the center through hole and the composite bone matrix gel (1). Step 5, drop the bone tumor or cartilage tumor seed cell suspension onto the composite bone matrix gel scaffold (1) in step 4, so that it fully infiltrates the composite bone matrix gel scaffold (1); Step 6, drop the complete culture solution into the bone tumor or cartilage tumor organ culture container (3) again, fully cover the bone matrix gel scaffold (1), and culture for 1-4 weeks to obtain a bone tumor or cartilage tumor organ.

6. The method for constructing cartilage organoids using the composite bone matrix gelatin scaffold according to claim 5, characterized in that, The cartilage seed cells to be inoculated in step 2 are selected from an exponentially growing human or animal-derived cartilage cell line or primary cartilage cells.

7. The method of claim 5, wherein the composite bone matrix gelatin scaffold is a porous scaffold having a pore size of 50-500 μιη, a porosity of 70-90%, and a thickness of 0.5-2 mm. The seeding amount of the chondrocyte seed cells to be inoculated in step 2 is 7 x 10 6 -10 x 10 6 cells per square millimeter of the composite bone matrix gelatin scaffold (1).

8. The method for constructing cartilage organoids using the composite bone matrix gelatin scaffold according to claim 5, characterized in that, After inoculating the cartilage seed cells in step 6, culture for 4 weeks.

9. The method of using the composite bone matrix gelatin scaffold to construct bone and cartilage tumor organoids according to any one of claims 1-4, wherein, The method comprises the following steps: Step 1, sterilize and disinfect the composite bone matrix gel scaffold (1) prepared according to any one of claims 1-4 for standby use; Step 2, resuspend the primary bone / primary cartilage tumor cells or bone / cartilage tumor cell lines to be inoculated in complete culture medium to obtain a cell suspension; Step 3, prepare a bone tumor or cartilage tumor organ culture container (3) and a flexible soft gasket (2); the shape and size of the culture container can be determined according to the purpose of generating a bone tumor or cartilage tumor organ; the flexible soft gasket (2) has upper and lower surfaces and a peripheral surface with a certain height, and the shape, size of the upper and lower surfaces, and height are adapted to the shape, caliber of the bone tumor or cartilage tumor bone organ culture container (3); the soft gasket can be placed in the bone tumor or cartilage tumor organ culture container (3), the radial size of the flexible soft gasket (2) is greater than the caliber size of the culture container (3), the flexible soft gasket (2) is tightly sealed with the inner wall of the culture container (3) by utilizing the elasticity of the flexible soft gasket (2), and is sealed at a required position in the bone tumor or cartilage tumor organ culture container (3); a through hole is made at the center point of the flexible soft gasket (2) according to the shape of the composite bone matrix gel (1), and the size of the through hole is smaller than that of the composite bone matrix gel (1); the bone tumor or cartilage tumor organ culture container (3) and the flexible soft gasket (2) are sterilized and disinfected for standby use; Step 4, press the composite bone matrix gel scaffold (1) for standby use in step 1 into the center through hole of the flexible soft gasket (2), utilize the elasticity of the center through hole to tightly seal the periphery of the through hole and the periphery of the composite bone matrix gel (1), and then seal the flexible soft gasket (2) at the required position in the bone tumor or cartilage tumor organ culture container (3) according to step 3; the liquid covering the composite bone matrix gel (1) cannot leak along the inner wall of the culture container (3) or seep out from the sealing position of the center through hole and the composite bone matrix gel (1); Step 5, drop the bone tumor or cartilage tumor seed cell suspension onto the composite bone matrix gel scaffold (1) in step 4, so that it fully infiltrates the composite bone matrix gel scaffold (1); Step 6, drop the complete culture solution into the bone tumor or cartilage tumor organ culture container (3) again, fully cover the composite bone matrix gel scaffold (1), and culture for 1-4 weeks to obtain a bone tumor or cartilage tumor organ.

10. The method of using the composite bone matrix gelatin scaffold to build bone and cartilage tumor organoids according to claim 9, wherein, The bone matrix gel scaffold in the composite bone matrix gel scaffold (1) in step 1 is selected from a scaffold material made of mammalian in-vitro cancellous bone.

11. The method of using the composite bone matrix gelatin scaffold to build bone and cartilage tumor organoids according to claim 9, wherein, The bone / cartilage tumor cells in step 2 are derived from mammals.

12. The method of claim 9, wherein the composite bone matrix gelatin scaffold is used to build bone and cartilage tumor organoids. The primary bone / tumor cells in step 2 are from the diseased side of the human or disease model animal, and are cultured in vitro.

13. The method of claim 9, wherein the composite bone matrix gelatin scaffold is used to build bone and cartilage tumor organoids. The bone tumor and cartilage tumor organoids in step 6 are cultured for 2 weeks.

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