CN118879620A - Preparation method and application of composite bone matrix gelatin scaffold - Google Patents

Abstract

The invention discloses a preparation method and application of a composite bone matrix gelatin stent, comprising the following steps: step 1, adding sterile water into the synthesized peptide gel powder, mixing to prepare an aqueous solution with 15% -40% of synthesized peptide substances, and detecting the pH value of the aqueous solution; step 2, placing the bone matrix gelatin scaffold prepared by the mammal in-vitro bone into the aqueous solution obtained in the step 1 to be fully soaked; step 3, adding sodium hydroxide solution into the aqueous solution in the step 2, adjusting the pH value of the aqueous solution to be 7-8, and stopping adding the sodium hydroxide solution; step 4, placing the gelatin stent soaked with the bone matrix obtained in the step 3 into an incubator, and observing the aqueous solution until the aqueous solution becomes a gel state, wherein the pH value of the aqueous solution is within the range of 7-8; and 5, taking out the bone matrix gelatin stent covered and filled with the synthetic peptide gel on the surface and in the pores in the incubator in the step 4, and freeze-drying to obtain the composite bone matrix gelatin stent.

Description

Preparation method and application of composite bone matrix gelatin scaffold

Technical Field

The invention relates to the field of biomedical technology, and in particular to a preparation method of a composite bone matrix gelatin scaffold and application thereof.

Background Art

Human organoids are miniaturized and simplified versions of human organs cultured in vitro. The 3D cell culture system 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 functions of real organs in vitro. This "minimal organ system" has great application potential in basic medical research such as organ development and homeostasis, as well as disease modeling, drug screening and personalized medicine.

In the process of constructing cartilage organoids, appropriate scaffold carriers are selected to mimic the biological structure and growth environment of natural cartilage tissue in order to generate stable cartilage organoids, that is, organoids that are consistent with the physiological tissue structure, cell differentiation type, and secretion of cartilage matrix of cartilage, and are applied to basic medical research such as cartilage differentiation and development and cartilage injury. However, tissue engineering cartilage technology similar to this technology is inconsistent with the actual problems solved and the purpose achieved by this technology. Tissue engineering cartilage technology is to inoculate chondrocytes onto degradable scaffold materials, and then implant them into the cartilage defect site. The implanted cells form new cartilage in the body to repair the defect site. Tissue engineering cartilage only requires that the inoculated chondrocytes can proliferate well on the scaffold and maintain their functions. It does not require the generation of cartilage morphological structures consistent with the physiological tissue structure in vitro.

Existing culture substrates for constructing cartilage organoids mostly use natural or synthetic extracellular matrix gel products (collectively referred to as matrix gel) to support cell growth. For example, matrix gel Micromass is used for 3D chondrocyte culture. However, the results only show that seed cells in different proliferation and differentiation states are accumulated in the matrix gel microspheres, and no cartilage tissue with a complete three-dimensional morphological structure is formed. The reason is that the matrix gel does not have a good three-dimensional porous structure, does not have a natural scaffold microenvironment for cartilage growth, and cannot generate a three-dimensional tissue structure consistent with the physiological structure. In patent documents such as CN 115645619A, CN1546654A, and CN106075580A, mammalian ex vivo bones are used to make natural bone matrix gelatin scaffolds (BMG for short) for preparing cartilage organoids. Although the bone matrix gelatin has a good three-dimensional porous structure and interconnected pores, its pore size ranges from 100-800 μm, which is too large for seed cells of 10 μm in size, resulting in low adhesion of the seed cells in the scaffold and easy loss, which cannot meet the basic conditions for the stable growth of cartilage organoids.

Tumor organoids are also a new type of preclinical model system for tumors. They can better reproduce the in vivo characteristics and heterogeneity of primary tumors and are currently an experimental model with extremely high reliability for modeling tumor diseases. Bone tumor and cartilage tumor organoids also face the problem of choosing suitable biological scaffold carriers when constructing them. In addition to the problems mentioned above in the culture process of cartilage organoids, such as the lack of three-dimensional porous structure of the scaffold carrier and the excessively large pore diameter, the problem of the source of multiple growth factors must also be solved. In the existing construction process of bone tumor and cartilage tumor organoids, in order to simulate the pathological microenvironment of highly proliferative tumor tissues in vivo, it is necessary to add exogenous growth factors of different types and compositions, chemical small molecule inhibitors/activators, etc., which makes the culture system complex and causes the culture price to be expensive and costly.

Due to the above main reasons, the culture methods of cartilage organoids, bone tumors and cartilage tumor organoids are immature, with low success rates and high costs. So far, there are few technical means to stably construct cartilage organoids with structural bionics and biofunctional bionics, and bone tumor and cartilage tumor organoids that reproduce the pathological characteristics of patient tumor tissues.

Summary of the invention

In order to overcome the shortcomings of the above-mentioned prior art, the present invention provides a method for preparing a composite bone matrix gelatin scaffold and the use of the same to construct cartilage organoids, bone tumors and cartilage tumor organoids, which can solve the problems that the existing scaffold materials do not have a three-dimensional structure with good pore size and porosity; the pore size is too large, resulting in a low seed cell adhesion rate; the addition of exogenous growth factors of different types and components leads to a complex system and high cost; the culture process is imprecise, and the above-mentioned organoids cannot be stably constructed.

The invention discloses a method for preparing a composite bone matrix gelatin scaffold material using synthetic peptide gel, comprising the following steps:

Synthetic peptide gel composite bone matrix gelatin scaffold:

Step 1, adding sterile water to synthetic peptide gel powder to prepare an aqueous solution containing 15% to 40% of synthetic peptide substances;

Step 2, placing the aqueous solution in a vortex oscillator, vortexing, oscillating, mixing evenly to form a mixed aqueous solution, and detecting the pH value thereof;

Step 3, placing the bone matrix gelatin scaffold prepared from mammalian ex vivo bone into the above-mentioned mixed aqueous solution and completely soaking it;

Step 4, gradually adding a small amount of sodium hydroxide solution to the mixed aqueous solution in step 3, adjusting the pH value of the mixed aqueous solution based on the measured pH value, and continuously measuring the pH value until it is within the range of 7-8 and then stopping the addition of sodium hydroxide solution;

Step 5, placing the mixed aqueous solution soaked with the bone matrix gelatin scaffold in step 4 with a pH value in the range of 7-8 in a 37° C. incubator, and observing it for 1-2 hours until it turns into a gel state;

Step 6, take out the bone matrix gelatin scaffold with the surface and pores covered with the synthetic peptide gel from the 37°C incubator, put it into a 4°C vacuum freeze dryer for 24-36 hours, and after freeze drying, obtain the composite bone matrix gelatin scaffold.

Preferably, the synthetic peptide gel is aldehyde-polyaspartic acid (molecular formula abbreviation: CHO-PASP) gel, methacrylamide-based gel, silk fibroin, etc., with aldehyde-polyaspartic acid (CHO-PASP) gel being preferred.

Preferably, the mammalian ex vivo bones in step 3 are selected from ex vivo bones of common experimental animals such as mice, rats, experimental rabbits, experimental miniature pigs, experimental dogs, experimental monkeys, etc. Considering the applicability and cost issues, ex vivo bones of experimental miniature pigs are preferred.

Preferably, the bone matrix gelatin scaffold prepared from mammalian ex vivo bone in step 3 above has a three-dimensional porous structure and is rich in a variety of growth factors that promote proliferation and differentiation, such as BMP, TGF-β1, IGF, FGF, etc., and the pore size is in the range of 100-700 μm.

Such synthetic peptide gel composite bone matrix gelatin scaffold not only maintains the three-dimensional porous structure of the original bone matrix gelatin scaffold, is conducive to the exchange of metabolic substances of seed cells, and is rich in a variety of factors that promote the proliferation and differentiation of seed cells and rapid growth, such as BMP, TGF-β1, IGF, FGF, etc., but also reduces the original bone matrix gelatin pore size of 100-700μm to a size of 10-20μm because the pore size is filled with synthetic peptide gel, providing the basic conditions and spatial environment for the stable growth of 10μm seed cells, improving the adhesion rate of seed cells, and reasonably distributing them to avoid loss. This solves the core problem of the inability to stably construct cartilage organoids, bone tumors, and cartilage tumor organoids.

The present invention also discloses an application method for constructing cartilage organoids based on the composite bone matrix gelatin scaffold, comprising the following steps:

Step 1, disinfecting and sterilizing the composite bone matrix gelatin scaffold prepared as described above for later use;

Step 2, resuspending the chondrocyte seed cells to be inoculated in a complete culture medium to obtain a chondrocyte suspension;

Step 3, prepare a cartilage organoid culture container and an elastic soft seal. The shape and size of the culture vessel can be determined according to the purpose of generating cartilage organoids; the elastic soft seal has an upper and lower surface and a peripheral surface of a certain height, and the shape, radial size of the upper and lower surfaces and height should be suitable for the shape and caliber size of the cartilage organoid culture container. The soft seal can be placed in the cartilage organoid culture container, and the caliber of the upper and lower surfaces of the soft seal is slightly larger than the caliber of the container, and the elasticity of the elastic soft seal is used to rub and seal tightly with the inner wall of the container, and seal it in the required position of the cartilage organoid culture container; a through hole is made at the center point of the elastic soft seal in 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 organoid culture container and the elastic soft seal are disinfected and sterilized for use;

Step 4, press the composite bone matrix gelatin scaffold prepared in step 1 into the central through hole of the elastic soft seal pad, so that the upper surface of the composite bone matrix gelatin is flush with the upper surface of the elastic soft seal pad. Since the central through hole is slightly smaller than the size of the composite bone matrix gelatin, the periphery of the through hole is tightly sealed with the periphery of the composite bone matrix gelatin scaffold by utilizing elasticity; then seal the elastic soft seal pad at the desired position in the cartilage organoid culture container as described in step 3; in this way, the liquid covering the composite bone matrix gelatin will not leak down along the inner wall of the culture container, nor will it seep out from the sealing portion between the central through hole and the composite bone matrix gelatin;

Step 5, slowly drop the cartilage seed cell suspension onto the composite bone matrix gelatin scaffold material in step 4, so that the composite bone matrix gelatin scaffold is fully infiltrated;

Step 6, add the complete culture medium dropwise into the cartilage organoid culture container to fully cover the composite bone matrix gelatin scaffold, and culture for 3 to 4 weeks to obtain cartilage organoids.

The biological characteristics of the cartilage seed cells selected by the present invention are adherent cells, which grow by attaching or adhering to the surface of the support. Therefore, the composite bone matrix gelatin scaffold of the present invention is sealed in a container to prevent the seed cells dripped on the composite bone matrix gelatin scaffold from leaking into the elastic soft seal pad and the container below the composite bone matrix gelatin scaffold, ensuring that the seed cells are fully infiltrated in the bone matrix gelatin scaffold and improving the attachment rate of the seed cells. This is one of the key culture links for stably constructing cartilage organoids and similar organoids that the present invention has explored and summarized in multiple experimental failures. We have tried to use biological glue such as agarose gel to seal the gap between the composite bone matrix gelatin scaffold and the cartilage organoid culture container, but agarose gel and the like will penetrate into the composite bone matrix gelatin scaffold and affect the entry and adhesion of the seed cells, making it impossible to stably construct cartilage organoids; we have also used a centrifuge tube with a conical lower part, cutting the composite bone matrix gelatin scaffold into a suitable size and using it to expand slightly after absorbing liquid and clamp it at the conical mouth of the lower part of the centrifuge tube, but due to the poor accuracy of manual cutting and the small elasticity of the bone matrix gelatin after absorbing liquid, although it can be clamped, it cannot achieve a sealed gap. Some seed cells still leak into the container below along the gap between the composite bone matrix gelatin scaffold and the inner wall of the cartilage organoid culture container, affecting the growth success rate of the organoids.

Preferably, the composite bone matrix gelatin scaffold described in step 1 can be cut into various shapes such as cylindrical, rectangular, cubic and other regular and irregular shapes according to the requirements of forming cartilage organoids, with cylindrical shape being preferred.

Preferably, the cartilage organoid culture container in step 3 can be a cylinder, a cone, a cylinder-cone mixture, etc., with a cylinder being preferred.

Preferably, the sealing methods of the cartilage organoid culture container, elastic soft seal, and composite bone matrix gelatin scaffold in step 3 and step 4 can refer to the several forms in Figure 12, so that the seed cells can fully infiltrate into the composite bone matrix gelatin scaffold under the action of gravity.

Preferably, in order to improve the efficiency and infiltration rate of seed cells infiltrating into the composite bone matrix gelatin scaffold material, the combination of the cartilage organoid culture container, the elastic soft seal, and the composite bone matrix gelatin in steps 3 and 4 can also generate negative pressure with the aid of the device in Figure 13 to compress and absorb the seed solution for full infiltration.

Preferably, the elastic soft seal in step 3 may be cylindrical, rectangular or in other shapes that match the cartilage organoid culture container, with cylindrical shape being preferred.

Preferably, the elastic soft sealing pad in step 3 can be made of transparent or opaque material, and the material can be selected from PVC soft rubber, silicone, polyurethane, etc., with transparent PVC soft rubber being preferred.

Preferably, the cartilage seed cells to be inoculated in step 2 are selected from primary chondrocytes or chondrocyte cell lines of human or animal origin in the exponential growth phase. At present, the technology closest to the present invention is the construction of tissue engineering cartilage technology for cartilage defect repair, but its inoculated cells mostly use bone marrow mesenchymal stem cells, adipose stem cells, etc., and it is necessary to add a variety of induction fluids with different components to induce stem cells to differentiate into chondrocytes. The present invention can preferably use primary chondrocytes or chondrocyte cell lines of human or animal origin in the exponential growth phase as seed cells, which are easy to obtain and can construct cartilage organoids in vitro without adding additional induction fluid. In addition, selecting seed cells in the exponential growth phase can ensure that the seed cells are good in vitality and promote the stable construction of cartilage organoids.

Preferably, the inoculation amount of the cartilage seed cells to be inoculated in step 2 is preferably 7×10 ⁶ -10×10 ⁶ per square millimeter of the composite bone matrix gelatin scaffold. The present invention has found in multiple experiments that the seed cell inoculation density is another key culture link that affects the stable construction of cartilage organoids, and the seed cells should maintain an appropriate inoculation number to obtain the best culture conditions. In the early stage, we used 1×10 ⁶ -6×10 ⁶ ATDC5 chondrocytes per 5mm (diameter) × 3mm (thickness) scaffold. After culture, only undifferentiated ATDC5 chondrocytes were accumulated in the pores of the scaffold, and no cartilage tissue with a physiological structure was formed (see Figure 5). This is because when the seed cell density is too low, rapid proliferation and differentiation cannot be carried out within a certain period of time, and there is a lack of interaction between cells, resulting in slow cell proliferation and differentiation. In addition, we used 11×10 ⁶ -12×10 ⁶ ATDC5 chondrocytes per 5mm (diameter) × 3mm (thickness) scaffold, and found that the seeded chondrocytes accumulated on the scaffold in large quantities, and no cartilage organoid tissue was formed after culture (see Figure 5). This is because the excessive number of seed cells, on the one hand, easily leads to cell aggregation to form cell clusters, which are difficult to enter the scaffold and cannot attach to the scaffold; on the other hand, under high density, the nutrients and space of the seed cells become limited, resulting in slow cell growth or even death, thereby hindering the construction of cartilage organoids.

Preferably, after the cartilage seed cells are inoculated in step 6, culture is performed for 3 to 4 weeks, preferably 4 weeks. The present invention has found in multiple experiments that after 1 week of culture, only the seed cells can be seen attached to the bone matrix gelatin pores, and after 2 weeks of culture, the seed cells in the bone matrix gelatin pores proliferate significantly, but no tissue structure is formed. After 3 weeks of culture, cartilage organoids consistent with the physiological structure are obtained, and after 4 weeks of culture, it can be seen that the obtained cartilage organoids have a more complete structure, neatly arranged cells, and stable tissue structure and biological function (Figure 6). After 5 weeks of culture, there was no obvious change in the structure of the new cartilage organoids.

In the embodiment of the present invention, the cartilage seed cells are preferably inoculated and cultured for 4 weeks, and the diameter of the generated cartilage organoids can reach 2-5 mm (Figure 4), which is much larger than the size of the cartilage organoids generated by the prior art. Therefore, it is more in line with the physiological structure and state of the human body, and has better beneficial effects for subsequent cartilage injury and differentiation research. The prior art mostly relies on the preparation of extracellular matrix gel products into microspheres as a culture matrix to construct organoids. Since the size of the microspheres themselves is too small, the diameter of the constructed organoids is often only 100-500 μm.

The present invention also discloses an application method for constructing bone tumor and cartilage tumor organoids based on the composite bone matrix gelatin scaffold, comprising the following steps:

Step 1, disinfecting and sterilizing the composite bone matrix gelatin scaffold for later use;

Step 2, resuspending the primary bone/chondrosarcoma cells or bone/chondrosarcoma cell lines to be inoculated in complete culture medium to obtain a cell suspension;

Step 3, prepare a bone tumor or cartilage tumor organoid culture container and an elastic soft seal. The shape and size of the culture vessel can be determined according to the purpose of generating bone tumor or cartilage tumor organoids; the elastic soft seal has an upper and lower surface and a peripheral surface of a certain height, and the shape, upper and lower surface size and height should be suitable for the shape and caliber of the bone tumor or cartilage tumor bone organoid culture container. The soft seal can be placed in the bone tumor or cartilage tumor organoid culture container, and the caliber of the upper and lower surfaces of the soft seal is slightly larger than the caliber of the container, and the elasticity of the elastic soft seal is used to rub and seal tightly with the inner wall of the container, and seal it 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 elastic soft seal in the shape of the composite bone matrix gelatin, and the size is slightly smaller than the size of the composite bone matrix gelatin. The bone tumor or cartilage tumor organoid culture container and the elastic soft seal should be disinfected and sterilized for use;

Step 4, pressing the composite bone matrix gelatin scaffold prepared in step 1 into the central through hole of the elastic soft seal pad, so that the upper surface of the composite bone matrix gelatin scaffold material is flush with the upper surface of the elastic soft seal pad, and at the same time utilizing the elasticity of the central through hole to tightly seal the periphery of the through hole with the periphery of the composite bone matrix gelatin, and then sealing the elastic soft seal pad at the desired position in the bone tumor or cartilage tumor organoid culture container as described in step 3, so that the liquid covering the composite bone matrix gelatin scaffold material will not leak down along the inner wall of the culture container, and will not seep out from the sealing portion between the central through hole and the composite bone matrix gelatin;

Step 5, adding the bone tumor or cartilage tumor seed cell suspension dropwise to the composite bone matrix gelatin scaffold in step 4, so that the composite bone matrix gelatin scaffold is completely infiltrated;

Step 6, add the complete culture medium dropwise into the bone tumor or cartilage tumor organoid culture container to fully cover the bone matrix gelatin scaffold, culture for 1-4 weeks, and obtain cartilage organoids.

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

Preferably, the bone/cartilage tumor cells in step 2 are from mammals.

Preferably, the primary bone/primary cartilage tumor cells in step 2 are obtained from the affected side of a subject with bone/cartilage tumor and cultured in vitro.

Preferably, the primary bone/cartilage tumor cells in step 2 are obtained from the affected side of a bone/cartilage tumor disease model animal and cultured in vitro.

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; the cartilage tumor cell lines include human chondrosarcoma cell lines SW1353, HCS-2/8, CAL-78 and mouse chondrosarcoma cell line EHS.

Preferably, the bone/cartilage tumor organoid culture time in step 6 is preferably 1-3 weeks, and optimally 2 weeks.

Compared with the prior art, the present invention has the following beneficial effects:

First, the application of the composite bone matrix gelatin combined with the key link technical method prepared by the present invention solves the technical problem that the prior art cannot stably construct cartilage organoids, bone tumors and cartilage tumor organoids. The cartilage organoids we constructed (see Figure 3), articular cartilage and epiphyseal cartilage have typical normal physiological structures, articular cartilage-like visible surface, middle and deep cartilage morphological structures, epiphyseal cartilage-like visible static layer, proliferation layer, hypertrophy layer and calcification layer, and even new bone trabeculae, have complete tissue structure and stable biological function, and simulate the physiological structure and function of human cartilage to the greatest extent. At the same time, the cartilage organoid cell diameter constructed by the present invention can reach 2-5mm (Fig. 4), which is much larger than the 100-500μm organoid constructed by the prior art, and is more in line with the physiological structure and state of the human body, and is applied to cartilage injury and differentiation research and basic medical research such as organ development, homeostasis and regeneration with better accuracy and precision. The bone tumor and cartilage tumor organoids we constructed are highly consistent with the characteristics of the original tumor tissues (Figures 8, 9, and 10). The diameter of the organoid cells can reach about 2 mm (see Figure 14), and they are highly consistent with the tissue structure, molecular characteristics, protein expression patterns, and drug responses of human origin. They have similar genome, transcriptome, morphology, and functional characteristics to patient tumor tissues, and can highly reproduce the original characteristics of in vivo tumors in vitro, providing an effective model for drug sensitivity research in tumor patients, precision targeted drug therapy, etc.

Second, the composite bone matrix gelatin scaffold prepared by the present invention retains the three-dimensional porous structure of bone matrix gelatin and a variety of growth factors such as BMP, TGF-β1, IGF, FGF, etc. that promote the proliferation and differentiation of organoids, and has excellent in vitro induction into organoids. It does not require the addition of additional growth factors, and can stably construct organoids using the corresponding seed cell conventional complete culture medium. It is simple, easy and low-cost. The pore size is reduced to the range of 10-20 μm, which meets the basic growth requirements of seed cells of about 10 μm in size, and overcomes the defect that the seed cells are difficult to attach due to the excessively large pore size of 100-700 μm, and are easy to lose and cannot stably construct organoids.

Third, the present invention solves the problems of seed cells not being fully infiltrated into the composite bone matrix gelatin scaffold and being lost, inappropriate seed cell inoculation concentration, inappropriate culture time, etc. It accurately grasps the key culture links, forms a mature culture process, and greatly improves the success rate of constructing cartilage organoids and bone/cartilage tumor organoids.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG1 is a scanning electron microscope image of the pore diameter of the aldehyde-polyaspartic acid (CHO-PASP) gel composite bone matrix gelatin scaffold;

FIG2 is a comparison of cartilage organoids generated by bone matrix gelatin scaffolds and cartilage organoids generated by aldehyde-polyaspartic acid (CHO-PASP) gel composite bone matrix gelatin scaffolds;

FIG3 is a staining image of articular cartilage organoids and epiphyseal cartilage organoids;

FIG4 is a diagram showing the diameter range of constructed articular cartilage organoids;

FIG5 is a graph showing the effect of seeding cell density on the formation of cartilage organoids;

Figure 6 shows the effect of different culture time on the construction of cartilage organoids;

Figure 7 is a diagram of bone tumor organoids;

FIG8 is a diagram of the histomorphological structure of bone tumor organoids;

FIG9 is a staining identification diagram of bone tumor organoids;

FIG10 is a diagram of the histomorphological structure of cartilage tumor organoids;

FIG11 is a diagram showing the method of using a cartilage/bone tumor/cartilage tumor organoid culture container;

FIG12 is a diagram showing the method of using a cartilage/bone tumor/cartilage tumor organoid culture container;

FIG13 is a diagram showing the method of using a cartilage/bone tumor/cartilage tumor organoid culture container;

FIG14 is a diagram showing the range of bone tumor organoid diameters;

FIG15 is a molecular structure diagram of aldehyde polyaspartic acid;

Figure numerals: 1 - composite bone matrix gelatin scaffold; 2 - elastic soft seal; 3 - culture container.

DETAILED DESCRIPTION

The present invention is further described in detail below with reference to Figures 1-15.

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

Example 1: Preparation method of composite bone matrix gelatin scaffold

Preparation method of synthetic peptide gel composite bone matrix gelatin scaffold:

0.3 g of sterile aldehyde-polyaspartic acid (CHO-PASP molecular structure is shown in FIG15 ) gel powder was weighed and placed in a 5 mL sterile centrifuge tube, and then 0.7 mL of sterile water was added thereto.

Use a vortex oscillator to vortex and shake, and observe to make it evenly mixed to form a CHO-PASP gel aqueous solution. Then use a pH test paper to detect the pH value of the CHO-PASP gel aqueous solution, which is about 4-5.

Then, a sterile bone matrix gelatin scaffold with a diameter of 5 mm and a thickness of 3 mm prepared from the cancellous bone of a miniature pig in vitro was immersed in the above-mentioned aldehyde polyaspartic acid (CHO-PASP) gel aqueous solution. After complete immersion, a sodium hydroxide solution with a concentration of 1 M was gradually added to the aldehyde polyaspartic acid (CHO-PASP) gel aqueous solution containing the bone matrix gelatin scaffold in small amounts, and the pH value of the aldehyde polyaspartic acid (CHO-PASP) gel aqueous solution was continuously detected during the period until the pH value was adjusted to 7.4 and 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 adjusted pH was placed in a 37°C incubator for observation, and the aqueous solution was observed to become a gel state after about 1 hour.

At this point, the CHO-PASP gel has covered the surface of the bone matrix gelatin and filled the pores in the bone matrix gelatin scaffold. The bone matrix gelatin complex is taken out with tweezers and placed in a 4°C vacuum freeze dryer for freeze drying for 24 hours to obtain a composite bone matrix gelatin scaffold.

2) Structural characteristics of composite bone matrix gelatin scaffold

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 by scanning electron microscopy. The results show that the composite bone matrix gelatin (BMG) scaffold filled with aldehyde polyaspartic acid (CHO-PASP) gel presents a three-dimensional porous structure with interconnected pores, as shown in Figure 1: the left picture shows the structure of the composite bone matrix gelatin under an electron microscope, and the right picture shows the distribution of pore sizes of the composite bone matrix gelatin scaffold, most of which are about 10-20μm in size.

The nutritional factors of the composite bone matrix gelatin scaffold were measured and are shown in Table 1.

Example 2: Preparation of articular cartilage organoids using composite bone matrix gelatin scaffolds

1) A method for constructing articular cartilage organoids using a composite bone matrix gelatin scaffold, comprising the following steps:

(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 in a clean bench for 48 hours (front and back sides for 24 hours each), and then soak it in PBS containing 100 U/mL penicillin and 100 μg/mL streptomycin for sterilization before use;

(2) C28/I2 chondrocytes in the exponential growth phase were routinely digested with trypsin and the cells were collected. The cells were resuspended in complete culture medium (DMEM/F12 culture medium containing 5% fetal bovine serum, 100 U/mL penicillin and 100 μg/mL streptomycin) and the concentration was adjusted to 8×10 ⁶ cells/mL.

(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 soft seal 2 with an upper and lower diameter of 8.55 mm and a thickness of 4 mm. A cylindrical through hole with a diameter of 4.9 mm and a radius of 2.45 mm from the center of the soft seal is chiseled out. The cylindrical round bottom centrifuge tube 3 and the cylindrical elastic soft seal 2 are sterilized and set aside for later use;

(4) The PBS covering the composite bone matrix gelatin scaffold 1 with a diameter of 5 mm and a thickness of 3 mm prepared in (1) is discarded, and the scaffold is taken out and pressed into the central through hole of the elastic soft seal 2, so that the upper surface of the composite bone matrix gelatin 1 is flush with the upper surface of the soft seal. The composite bone matrix gelatin scaffold 1 with a diameter of 5 mm and a thickness of 3 mm is tightly sealed in the through hole of the soft seal with a diameter of 4.9 mm and a thickness of 4 mm due to its elasticity; then the cylindrical elastic soft seal 2 with a diameter of 8.55 mm and a thickness of 4 mm sealed with the composite bone matrix gelatin scaffold 1 is evenly pushed to a position 3 mm away from the bottom of a 5 mL cylindrical round-bottom centrifuge tube 3 with a diameter of 8.5 mm and a height of 41 mm. The elastic 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 due to its elasticity; as shown in FIG. 11

(5) Take 1 mL of the prepared C28/I2 chondrocyte suspension and slowly drip it vertically onto the composite bone matrix gelatin scaffold 1 at the bottom of the centrifuge tube, let it stand for 10 minutes, and observe that it is fully infiltrated into the composite bone matrix scaffold material 1;

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

(7) After the culture is completed, the culture medium is discarded, and the elastic soft seal 2 is evenly pulled out of the cylindrical round-bottom centrifuge tube 3 with force, and the tissue block in the central through hole of the elastic soft seal 2 is taken out to obtain the articular cartilage organoid.

The left side of Figure 2 shows a cartilage organoid generated using a bone matrix gelatin scaffold, and the right side shows a cartilage organoid generated using a composite bone matrix gelatin scaffold 1. The cartilage organoid on the right side has obviously denser arrangement of chondrocytes than the left one, with obvious cartilage pits, rich extracellular matrix, and has the typical scattered cell arrangement characteristics of cartilage.

2) Identify the histomorphological structure of articular cartilage organoids.

The articular cartilage organoids obtained by culture in Example 2 were fixed with 4% paraformaldehyde for 30 minutes, dehydrated with conventional gradient alcohol, and then embedded in wax blocks for later use.

5 μm paraffin sections were prepared, and after conventional dewaxing and dehydration, conventional H&E staining and TB staining, they were placed under an optical microscope for observation and photography, as shown in Figure 3A. The results showed that in the constructed articular cartilage organoids, the newly formed surface chondrocytes were flat, the middle chondrocytes were oval or round, the deep chondrocytes were hypertrophic, and the layers were arranged neatly and orderly, forming a surface, middle and deep structure consistent with physiological articular cartilage, with obvious different differentiation stages and zonal characteristics. The TB staining results showed that the extracellular matrix of the constructed articular cartilage organoids was bluish purple and evenly distributed, indicating that the articular cartilage organoids constructed by the present invention have the characteristics of normal cartilage matrix secretion. Figures 4a and b show that the diameter of the constructed articular cartilage organoids ranges from about 2 to 5 mm.

Example 3: Construction of epiphyseal cartilage organoids using composite bone matrix gelatin scaffold 1

1) A method for constructing epiphyseal cartilage organoids using the composite bone matrix gelatin scaffold 1, comprising the following steps:

(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 in a clean bench for 48 hours (front and back sides for 24 hours each), and then soak it in PBS containing 100 U/mL penicillin and 100 μg/mL streptomycin for sterilization before use;

(2) Take ATDC5 chondrocytes in the exponential growth phase, digest them 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) and adjust the concentration to 9×10 ⁶ cells/mL.

(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 soft seal 2 with an upper and lower diameter of 8.55 mm and a thickness of 4 mm. A cylindrical through hole with a diameter of 4.9 mm is chiseled out with a radius of 2.45 mm from the center of the soft seal. The cylindrical round bottom centrifuge tube 3 and the cylindrical elastic soft seal 2 are sterilized and set aside for later use;

(4) The PBS covering the composite bone matrix gelatin scaffold 1 with a diameter of 5 mm and a thickness of 3 mm prepared in (1) is discarded, and the composite bone matrix gelatin scaffold 1 is taken out and pressed into the central through hole of the elastic soft seal 2, so that the upper surface of the composite bone matrix gelatin scaffold 1 is flush with the upper surface of the soft seal. The composite bone matrix gelatin scaffold 1 with a diameter of 5 mm and a thickness of 3 mm is tightly sealed in the through hole of the soft seal with a diameter of 4.9 mm and a thickness of 4 mm due to its elasticity; then the cylindrical elastic soft seal 2 with a diameter of 8.55 mm and a thickness of 4 mm sealed with the composite bone matrix gelatin scaffold 1 is evenly pushed to a position 3 mm away from the bottom of a 5 mL cylindrical round-bottom centrifuge tube 3 with a diameter of 8.5 mm and a height of 41 mm. The elastic soft seal 2 with a diameter of 8.55 mm is tightly sealed in the centrifuge tube with a diameter of 8.5 mm due to its elasticity, as shown in FIG. 11 ;

(5) Take 1 mL of the prepared ATDC5 chondrocyte suspension and slowly drip it vertically onto the composite bone matrix gelatin scaffold 1 at the bottom of the centrifuge tube, let it stand for 10 minutes, and observe that it is fully infiltrated into the scaffold;

(6) 3 mL of complete culture medium was added dropwise into the round-bottom centrifuge tube 3 to fully cover the composite bone matrix gelatin scaffold 1, and the tube was placed in an incubator for culture. The culture medium was replaced every two days for 4 weeks.

(7) After the culture is completed, the culture medium is discarded, and the elastic soft seal 2 is evenly pulled out of the cylindrical round-bottom centrifuge tube 3 with force, and the tissue block in the central through hole of the elastic soft seal 2 is taken out to obtain the epiphyseal cartilage organoid tissue.

2) Identify the histomorphological structure of epiphyseal cartilage organoids.

(1) The epiphyseal cartilage organoid tissue obtained by culture in Example 3 was fixed with 4% paraformaldehyde for 30 minutes, dehydrated with conventional gradient alcohol, and then embedded in wax blocks for later use.

(2) 5 μm paraffin sections were prepared, and after routine dewaxing and dehydration, they were stained with H&E and TB, and then placed under an optical microscope for observation and photography, as shown in Figure 3B. The results showed that the H&E staining results showed that the newly formed chondrocytes in the epiphyseal cartilage organoids proliferated significantly and were oval or round in shape. The chondrocytes were arranged neatly and orderly, forming a columnar arrangement structure close to the normal epiphyseal cartilage in vivo, with obvious characteristics of different differentiation stages and zones. The cells in the upper layer of the newly formed tissue of the epiphyseal cartilage organoids were smaller in size and less in number, which was close to the chondrocytes in the resting layer of the epiphyseal cartilage in vivo; the cells in the middle layer proliferated actively, were oval in shape, larger in size, and arranged in a longitudinal columnar shape, which was close to the chondrocytes in the proliferative layer of the epiphyseal cartilage in vivo; while the cells in the deep layer became larger in size, the cell bodies were rounded, and arranged in a columnar longitudinal shape, which was close to the chondrocytes in the proliferative layer of the epiphyseal cartilage in vivo. The TB staining results showed that the extracellular matrix of the epiphyseal cartilage organoids was evenly distributed in blue-purple, and the matrix around the deep mast cells was darker, indicating that the epiphyseal cartilage had the secretory characteristics of cartilage tissue. Figure 4c and d show that the diameter of the constructed epiphyseal cartilage organoids ranged from about 2mm to 5mm, reaching the millimeter level.

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

1. Use composite bone matrix gelatin scaffold 1 to prepare osteosarcoma organoids.

1) Select the 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 (front and back sides for 24 hours each) in a clean bench, and then soak it in PBS containing 100 U/mL penicillin and 100 μg/mL streptomycin for sterilization before use;

2) Human osteosarcoma U2OS cell line in logarithmic growth phase was obtained, digested with trypsin and collected, and the cells were resuspended in McCoy's 5A complete medium (containing 10% fetal bovine serum, 100 U/mL penicillin and 100 μg/mL streptomycin) to adjust the concentration to 8×10 ⁶ cells/mL.

3) Prepare a 5mL cylindrical round-bottom glass centrifuge tube 3 with a diameter of 8.5mm and a height of 41mm and a cylindrical elastic transparent silicone soft seal 2 with an upper and lower diameter of 8.55mm and a thickness of 4mm. A cylindrical through hole with a diameter of 4.9mm is chiseled with a radius of 2.45mm from the center of the soft seal. The cylindrical round-bottom centrifuge tube 3 and the cylindrical elastic soft seal 2 are sterilized and set aside for use;

4) The PBS covering the composite bone matrix gelatin scaffold 1 with a diameter of 5 mm and a thickness of 3 mm prepared in (1) is discarded, and the scaffold is taken out and pressed into the central through hole of the elastic soft seal 2, so that the upper surface of the composite bone matrix gelatin scaffold 1 is flush with the upper surface of the soft seal. The composite bone matrix gelatin scaffold 1 with a diameter of 5 mm and a thickness of 3 mm is tightly sealed in the through hole of the soft seal with a diameter of 4.9 mm and a thickness of 4 mm due to its elasticity; then the cylindrical elastic soft seal 2 with a diameter of 8.55 mm and a thickness of 4 mm sealed with the composite bone matrix gelatin scaffold 1 is evenly pushed to a position 3 mm away from the bottom of a 5 mL cylindrical round-bottom centrifuge tube 3 with a diameter of 8.5 mm and a height of 41 mm. The elastic 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 due to its elasticity; as shown in FIG. 11 .

5) Take 1 mL of U2OS cell suspension and slowly drop it vertically onto the composite bone matrix gelatin scaffold 1 material at the bottom of centrifuge tube 3, let it stand for 10 minutes, and observe that it is fully infiltrated into the scaffold;

6) Add 3 mL of McCoy's 5A complete medium and culture in a 37°C, 5% CO2 incubator for 2 weeks, replacing fresh medium every other day. After the culture is completed, discard the culture medium, pull the elastic soft seal 2 out of the centrifuge tube 3 with uniform force, and take out the tissue block in the central through hole of the elastic soft seal 2 to obtain the osteosarcoma organoid. As shown in Figure 7.

2. Identification of the histomorphological structure of osteosarcoma organoids

1) The osteosarcoma organoids obtained by culture in step 1 of Example 4 were fixed with 4% paraformaldehyde for 30 minutes, dehydrated in a conventional gradient and embedded in wax blocks. 5 μm paraffin sections were prepared, dewaxed and hydrated in a conventional manner, and then stained with hematoxylin and eosin. After staining, the sections were dehydrated and sealed;

The osteosarcoma organoids obtained by culture were fixed with 4% paraformaldehyde for 30 minutes, dehydrated in a conventional gradient and embedded in wax blocks. 5 μm paraffin sections were prepared, and after conventional dewaxing and hydration, H&E, Masson, Sirius red staining, and ALP and OCN immunohistochemical staining were performed, and the sections were dehydrated and sealed after staining.

2) Take photos and observe under an optical microscope, as shown in Figure 8a. The microscope shows that osteosarcoma organoid tissue structures can be formed on the bone matrix gelatin 1 scaffold and in the pores of the scaffold. The tumor cells are irregular in shape, with significant atypia and polymorphism, abnormally enlarged nuclei, and easy to see nuclear division images; secondly, a small amount of irregular grid-like bone-like tissue and woven bone are formed between the tumor cells. These tissue structures are consistent with the tumor morphology of osteosarcoma patients (as shown in Figure 8b), indicating that osteosarcoma organoids have been successfully and stably constructed based on the present invention.

Photographs were taken under an optical microscope, as shown in Figure 9. Masson staining dyed new osteocytes blue and mature osteocytes blue (as shown in Figure 9a); Sirius red staining dyed osteocytes red to show collagen fiber generation (as shown in Figure 9b); ALP and OCN immunohistochemical staining dyed osteoblasts brown (as shown in Figure 9c).

The above identifications all indicate that the constructed organoids are osteosarcoma organoids.

3. Preparation of Chondrosarcoma Organoids Using Composite Bone Matrix Gelatin Scaffold 1

1) Select the 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 (front and back sides for 24 hours each) in a clean bench, and then soak it in PBS containing 100 U/mL penicillin and 100 μg/mL streptomycin for sterilization before use;

2) Human chondrosarcoma SW1353 cell line in logarithmic growth phase was obtained, and the cells were routinely digested with trypsin and collected. The cells were resuspended in a special culture medium for SW1353 cells (L15) (containing 10% fetal bovine serum, 100 U/mL penicillin and 100 μg/mL streptomycin) and the concentration was adjusted to 9×10 ⁶ cells/mL.

3) Prepare a 5mL cylindrical round-bottom glass centrifuge tube 3 with a diameter of 8.5mm and a height of 41mm and a cylindrical elastic transparent silicone soft seal 2 with an upper and lower diameter of 8.55mm and a thickness of 4mm. A cylindrical through hole with a diameter of 4.9mm is chiseled with a radius of 2.45mm from the center of the soft seal. The cylindrical round-bottom centrifuge tube 3 and the cylindrical elastic soft seal 2 are sterilized and set aside for use;

4) The PBS covering the composite bone matrix gelatin scaffold 1 with a diameter of 5 mm and a thickness of 3 mm prepared in 1) is discarded by suction, and the PBS is taken out and pressed into the central through hole of the elastic soft seal 2, so that the upper surface of the composite bone matrix gelatin scaffold 1 is flush with the upper surface of the soft seal. 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 seal through hole with a diameter of 4.9 mm and a thickness of 4 mm due to its elasticity; then the cylindrical elastic soft seal 2 with a diameter of 8.55 mm and a thickness of 4 mm sealed with the composite bone matrix gelatin scaffold 1 is evenly pushed to a place 3 mm away from the bottom of a 5 mL cylindrical round-bottom centrifuge tube 3 with a diameter of 8.5 mm and a height of 41 mm. The elastic soft seal 2 with a diameter of 8.55 mm is tightly blocked in the centrifuge tube 3 with a diameter of 8.5 mm due to its elasticity, as shown in Figure 11;

5) Take 1 mL of SW1353 cell suspension and slowly drop it vertically onto the composite bone matrix gelatin scaffold 1 at the bottom of centrifuge tube 3, let it stand for 10 minutes, and observe that it is fully infiltrated into the scaffold;

6) Add 4 mL of SW1353 cell-specific culture medium (L15) and culture in a 37°C, 5% CO2 incubator for 2 weeks, replacing fresh culture medium every other day. After the culture is completed, discard the culture medium, evenly pull the elastic soft seal 2 out of the cylindrical round-bottom centrifuge tube 3 with force, and take out the tissue block in the central through hole of the elastic soft seal 2 to obtain the chondrosarcoma organoid. As shown in Figure 10a.

4. Identification of chondrosarcoma organoid tissue morphology

1) The chondrosarcoma organoids obtained by culture in step 3 of Example 4 were fixed with 4% paraformaldehyde for 30 minutes, dehydrated in a conventional gradient and embedded in wax blocks. 5 μm paraffin sections were prepared, dewaxed and hydrated in a conventional manner, and then stained with hematoxylin and eosin. After staining, the sections were dehydrated and sealed.

2) Photographing and observation under an optical microscope, as shown in FIG10a, shows that chondrosarcoma organoid-like tissue structures can be formed on the composite bone matrix gelatin scaffold 1 and in the pores of the scaffold, with abundant tumor cells, nuclei of different sizes, irregular dark staining, binuclei, multinuclei, atypical nuclei and nuclear division images, abundant cartilage matrix, often accompanied by calcification and ossification. The constructed chondrosarcoma organoids are consistent with the morphological structure of chondrosarcoma patient tissues (as shown in FIG10b), indicating that the constructed organoids are chondrosarcoma organoids.

This specific embodiment is merely an explanation of the invention and is not a limitation of the invention. After reading this specification, those skilled in the art may make non-creative modifications to this embodiment as needed. However, as long as they are within the scope of protection of the invention, they are protected by patent law.

Table 1 Detection of growth factors related to the protein profile of bone matrix gelatin 1 scaffold

Claims (13)

1. A method for preparing a composite bone matrix gelatin scaffold, characterized in that it comprises the following steps: Step 1, mixing the synthetic peptide gel powder with sterile water to prepare an aqueous solution containing 15% to 40% of the synthetic peptide substance, and detecting the pH value thereof; Step 2, placing the bone matrix gelatin scaffold prepared from mammalian ex vivo bone into the aqueous solution obtained in step 1 and fully soaking it; Step 3, gradually adding sodium hydroxide solution to the aqueous solution of step 2, adjusting the pH value of the aqueous solution until the pH value is in the range of 7-8, and stopping adding sodium hydroxide solution; Step 4, placing the aqueous solution soaked with the bone matrix gelatin scaffold obtained in step 3 and having a pH value in the range of 7-8 into an incubator, and observing the aqueous solution until it becomes a gel state; Step 5, taking out the bone matrix gelatin scaffold covered with and filled with synthetic peptide gel on the surface and in the pores in the incubator in step 4, and freeze-drying it to obtain a composite bone matrix gelatin scaffold (1). 2. The method for preparing a composite bone matrix gelatin scaffold according to claim 1, characterized in that the synthetic peptide gel in step 1 comprises: aldehyde-based polyaspartic acid gel or methacrylamide-based gel or silk fibroin. 3. The method for preparing a composite bone matrix gelatin scaffold according to claim 1, characterized in that the mammalian ex vivo bone in step 2 is an ex vivo bone of a mouse, a rat, an experimental rabbit, an experimental miniature pig, an experimental dog, or an experimental monkey. 4. The method for preparing a composite bone matrix gelatin scaffold according to claim 1, characterized in that the pore size range of the bone matrix gelatin scaffold prepared from mammalian ex vivo bone in step 2 is 100-700 μm. 5. The method for constructing cartilage organoids using the composite bone matrix gelatin scaffold according to any one of claims 1 to 4, characterized in that it comprises the following steps: Step 1, disinfecting and sterilizing the composite bone matrix gelatin scaffold (1) prepared according to any one of claims 1 to 4 for later use; Step 2, resuspending the chondrocyte seed cells to be inoculated in a complete culture medium to obtain a chondrocyte suspension; Step 3, preparing a cartilage organoid culture container (3) and an elastic soft seal (2); the shape and size of the culture container can be determined according to the purpose of generating cartilage organoids; the elastic soft seal (2) has an upper surface, a lower surface and a peripheral surface of a certain height, and the shape, upper and lower surface sizes and height are adapted to the shape and caliber of the cartilage organoid culture container (3); the elastic soft seal (2) can be placed in the cartilage organoid culture container (3), the radial size of the elastic soft seal (2) is larger than the caliber of the culture container (3), and the elastic soft seal (2) is used to frictionally seal with the inner wall of the culture container (3) to seal at the required position of the cartilage organoid culture container (3); a through hole is made at the center point of the elastic soft seal (2) in the shape of the composite bone matrix gelatin (1), and the size of the through hole is smaller than the size of the composite bone matrix gelatin (1); the cartilage organoid culture container (3) and the elastic soft seal (2) are disinfected and sterilized for use; Step 4, pressing the composite bone matrix gelatin scaffold (1) prepared in step 1 into the central through hole of the elastic soft seal (2), using the elasticity of the central through hole to tightly seal the periphery of the through hole with the periphery of the composite bone matrix gelatin (1), and then sealing the elastic soft seal (2) at the desired position in the cartilage organoid culture container (3) as described in step 3, so that the liquid covering the composite bone matrix gelatin (1) will not leak down along the inner wall of the culture container (3), and will not seep out from the sealing portion between the central through hole and the composite bone matrix gelatin (1); Step 5, adding the cartilage seed cell suspension dropwise onto the composite bone matrix gelatin scaffold (1) in step 4, so that the suspension fully permeates the composite bone matrix gelatin scaffold (1); Step 6, add the complete culture solution dropwise into the cartilage organoid culture container (3) to fully cover the bone matrix gelatin scaffold (1), and culture for 3 to 4 weeks to obtain cartilage organoids. 6. The application method of the composite bone matrix gelatin scaffold for constructing cartilage organoids according to claim 5, characterized in that the cartilage seed cells to be inoculated in step 2 are selected from human or animal-derived chondrocyte lines or primary chondrocytes in the exponential growth phase. 7. The application method of the composite bone matrix gelatin scaffold for constructing cartilage organoids according to claim 5, characterized in that the inoculation amount of the cartilage seed cells to be inoculated in step 2 is 7×10 ⁶ -10×10 ⁶ per square millimeter of the composite bone matrix gelatin scaffold (1). 8. The application method of the composite bone matrix gelatin scaffold for constructing cartilage organoids according to claim 5, characterized in that after the cartilage seed cells are inoculated in step 6, they are cultured for 4 weeks. 9. The method for using the composite bone matrix gelatin scaffold to construct bone tumor and cartilage tumor organoids according to any one of claims 1 to 4, characterized in that it comprises the following steps: Step 1, disinfecting and sterilizing the composite bone matrix gelatin scaffold (1) prepared according to any one of claims 1 to 4 for later use; Step 2, resuspending 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 organoid culture container (3) and an elastic soft seal (2); the shape and size of the culture container can be determined according to the purpose of generating bone tumor or cartilage tumor organoid; the elastic soft seal (2) has an upper and lower surface and a peripheral surface of a certain height, and the shape, upper and lower surface size and height are adapted to the shape and caliber of the bone tumor or cartilage tumor bone organoid culture container (3); the soft seal can be placed in the bone tumor or cartilage tumor organoid culture container (3), the radial dimension of the elastic soft seal (2) is larger than the caliber of the culture container (3), and the elasticity of the elastic soft seal (2) is used to frictionally seal with the inner wall of the culture container (3) and seal it in the required position of the bone tumor or cartilage tumor organoid culture container (3); a through hole is made at the center point of the elastic soft seal (2) in the shape of the composite bone matrix gelatin (1), and the size of the through hole is smaller than the size of the composite bone matrix gelatin (1); the bone tumor or cartilage tumor organoid culture container (3) and the elastic soft seal (2) are sterilized and ready for use; Step 4, pressing the composite bone matrix gelatin scaffold (1) prepared in step 1 into the central through hole of the elastic soft seal (2), using the elasticity of the central through hole to tightly seal the periphery of the through hole with the periphery of the composite bone matrix gelatin (1), and then sealing the elastic soft seal (2) at the desired position in the bone tumor or cartilage tumor organoid culture container (3) as described in step 3; so that the liquid covering the composite bone matrix gelatin (1) will not leak down along the inner wall of the culture container (3), and will not seep out from the sealing portion between the central through hole and the composite bone matrix gelatin (1); Step 5, adding the bone tumor or cartilage tumor seed cell suspension dropwise onto the composite bone matrix gelatin scaffold (1) in step 4, so that the suspension fully permeates the composite bone matrix gelatin scaffold (1); Step 6, add the complete culture solution dropwise into the bone tumor or cartilage tumor organoid culture container (3) to fully cover the composite bone matrix gelatin scaffold (1), and culture for 1-4 weeks to obtain the bone tumor or cartilage tumor organoid. 10. The application method of the composite bone matrix gelatin scaffold for constructing bone tumor and cartilage tumor organoids according to claim 9, characterized in that the bone matrix gelatin scaffold in the composite bone matrix gelatin scaffold (1) in step 1 is a scaffold material made of mammalian ex vivo cancellous bone. 11. The method for using the composite bone matrix gelatin scaffold to construct bone tumor and cartilage tumor organoids according to claim 9, characterized in that the bone/cartilage tumor cells in step 2 are from mammals. 12. The application method of the composite bone matrix gelatin scaffold for constructing bone tumor and cartilage tumor organoids according to claim 9, characterized in that the primary bone/primary cartilage tumor cells in step 2 are obtained from the affected side of humans or disease model animals and cultured in vitro. 13. The application method of the composite bone matrix gelatin scaffold for constructing bone tumor and cartilage tumor organoids according to claim 9, characterized in that the culture time of the bone tumor and cartilage tumor organoids in step 6 is 2 weeks.