CN117530814A - A skeletal organoid construction system and its use method - Google Patents

Abstract

The invention discloses a skeletal organoid construction system and its use method. The construction system includes the following modules: a bone defect data module, which is used to obtain the volumetric inspection data of the bone defect area of the patient obtained through medical imaging technology, and obtain the patient's bone defect data module. Medical imaging data of the defect area; a three-dimensional simulation module, used to convert the acquired medical imaging data into a three-dimensional image, and perform a three-dimensional simulation of the patient's bone defect area; generate three-dimensional data of the skeletal organoids to be constructed based on the three-dimensional image of the bone defect area Model; a three-dimensional building module. According to the three-dimensional data model of the skeletal organoid to be constructed, a suitable biocompatible scaffold is selected, and the cultured osteoblast layer is planted on the biocompatible scaffold to allow it to grow on the bone microstructure. Growing in the environment, the osteoblast layer grows and forms skeletal organoids. The present invention can construct and form skeletal organoids that match the shape and structure of the bone defect area to better complete clinical treatment.

Description

A skeletal organoid construction system and its use method

Technical field

The present invention relates to the technical field of skeletal organoids, and in particular to a skeletal organoid construction system and a method of using the same.

Background technique

Organoids are tiny cellular structures that simulate the anatomy and function of human organs. Such organ models grown in the laboratory play an increasingly important role in modern medical research. Organoid technology can take advantage of the self-organizing properties of mammalian pluripotent stem cells or stem cells derived from adult tissues to construct a 3D microenvironment in vitro similar to that in vivo, and construct organoids with multiple cell types. These organoids with the complexity of multiple cell types have been It is highly similar to the corresponding tissues and organs in the body, providing an ideal platform for studying the development, regeneration and pathology of tissues and organs.

In orthopedics, a large number of people suffer from bone defects every year due to trauma, inflammation, tumor resection and other reasons. However, the human body itself cannot regenerate and repair large critical bone defects. In most cases, external surgical intervention is required to restore normalcy. Currently, the commonly used bone graft materials in clinical practice include the following: Autologous bone, that is, the material is taken from the patient’s own body. It is the most ideal repair material, but it suffers from secondary injuries, complications and sources of multiple surgical and donor sites. Problems such as insufficiency; allogeneic bone, mostly from cadaver donations or animals, has issues such as immune response, potential infection risks, and medical ethics; bone-filled prostheses have rejection reactions, poor bone ingrowth success rates, and require revision after a long period of time And other issues. Therefore, using organoid technology to construct organoids that are highly similar to skeletal organs in the body can better fulfill the task of clinical treatment.

However, in organoid technology, organoids that simulate the brain, lungs and other organs have been available for a long time, but creating organoid models for bone tissue has been very difficult. For example, the Chinese patent application with publication number CN115154674A discloses "a 3D bioprinted bone-like tissue engineering scaffold based on bone organoids." This application directly encapsulates cells in the scaffold during the printing process, allowing for high-throughput manufacturing. , and precisely control cells. However, this application is prone to differentiation and destruction of cells during the 3D printing process, causing distortion of the constructed skeletal organoids. It is difficult to construct the required skeletal organoids because different types of bones Cells exist in a special extracellular matrix (ECM), which is a network composed of collagen and minerals. They are always in a state of constant change, so the structure is consistent with the bone defect area. Adapting skeletal organoids is more difficult.

Contents of the invention

In order to solve the technical problem in the prior art that it is difficult to construct skeletal organoids that are adapted to bone defect areas, the present invention provides the following technical solutions.

A skeletal organoid construction system of the present invention includes the following modules:

The bone defect data module is used to obtain the volumetric examination data of the patient's bone defect area obtained through medical imaging technology, and obtain the medical imaging data of the patient's bone defect area;

The three-dimensional simulation module is used to convert the acquired medical imaging data into a three-dimensional image and perform a three-dimensional simulation of the patient's bone defect area; generate a three-dimensional data model of the skeletal organoid to be constructed based on the three-dimensional image of the bone defect area;

The three-dimensional building module selects an appropriate biocompatible scaffold based on the three-dimensional data model of the skeletal organoid to be constructed, and plants the cultured osteoblast layer on the biocompatible scaffold to allow it to grow in the bone microenvironment. Growth, over time, the osteoblast layer grows and forms skeletal organoids.

As a further technical solution, the medical imaging data includes one or more of X-ray imaging, ultrasound imaging, computed tomography CT, magnetic resonance imaging MRI, positron emission computed tomography PET-CT, etc. .

As a further technical solution, in the process of converting the acquired medical imaging data into a three-dimensional image, the original three-dimensional image of the bone defect area is restored and compared with the three-dimensional image of the defect part corresponding to the defect area.

As a further technical solution, the three-dimensional building module also includes a microenvironment unit that provides a growth environment for the osteoblast layer, a planting unit that implants the osteoblast layer on a biocompatible scaffold, and a continuous construction unit that is suitable for the bone defect area. The building blocks of skeletal organoids.

As a further technical solution, the microenvironment unit is used to create a microenvironment for the growth of osteoblasts, including providing cells related to bone tissue formation and the environment required for the growth of skeletal organoids to be constructed, while cultivating osteoblasts layers and three-dimensional bone tissue.

As a further technical solution, the cells related to bone tissue formation include at least one or more cells such as bone marrow stromal cells, osteoprogenitor cells, pre-osteoblasts, osteoblasts, bone lining cells, osteocytes or osteoclasts. combination of cells.

As a further technical solution, the structure of the biocompatible scaffold is determined based on the actual detected bone defect area in the planting unit, and the cultured osteoblast layer is planted on the biocompatible scaffold to promote the growth of osteoblasts. Cells align or stretch in specific directions, allowing them to grow in an osteogenic microenvironment.

As a further technical solution, the construction unit is used to continuously construct skeletal organoids that are adapted to the bone defect area. According to the different structures and microenvironments of different skeletal organoids, mechanical force is used to simulate the pressure required for human bone formation. The bone marrow stem cells are transformed into a variety of osteoblasts such as osteoblasts and growth-regulating osteocytes required for the growth of the skeletal organoids to be constructed, and further form skeletal organoids that match the shape and structure of the bone defect area.

The present invention also includes a method for using a skeletal organoid construction system, which includes the following steps:

S1: Detect the patient's bone defect area and obtain the volumetric examination data of the patient's bone defect area obtained through medical imaging technology;

S2: Use computer 3D auxiliary software to convert the acquired medical imaging data into 3D images, perform 3D simulation of the patient's bone defect area, and generate a 3D data model of the skeletal organoids to be constructed;

S3: Select an appropriate biocompatible scaffold based on the three-dimensional data model of the skeletal organoid to be constructed, and plant the cultured osteoblast layer on the biocompatible scaffold to allow it to grow in the bone microenvironment. Over time, the osteoblast layer grows and forms skeletal organoids.

As a further technical solution, step S3 also includes step S301, using human stem cells to create a microenvironment for the growth of osteoblasts, that is, creating cells related to bone tissue formation, and simultaneously cultivating the osteoblast layer and three-dimensional bone tissue;

It also includes step S302 of planting the cultured osteoblast layer on a biocompatible scaffold adapted to the bone defect area to form a construct;

It also includes step S303, which continues to construct skeletal organoids that are suitable for the bone defect area. According to the different structures and microenvironments of different skeletal organoids, mechanical force is used to simulate the pressure required for human bone formation to convert bone marrow stem cells into the required The growth of constructed skeletal organoids requires a variety of osteoblasts such as osteoblasts and growth-regulating osteocytes.

The beneficial effects of the present invention are that the present invention obtains three-dimensional data of the patient's bone defect area through medical imaging technology, converts the obtained medical image data into a three-dimensional image, performs a three-dimensional simulation of the patient's bone defect area, and generates data based on the three-dimensional image of the bone defect area. A three-dimensional data model of the skeletal organoid to be constructed; then, according to the three-dimensional data model of the skeletal organoid to be constructed, a suitable biocompatible scaffold is selected, and the cultured osteoblast layer is planted on the biocompatible scaffold According to the different structures and microenvironments of different skeletal organoids, mechanical force is used to simulate the pressure required for human bone formation, so that bone marrow stem cells are transformed into osteoblasts and growth-regulating osteocytes required for the growth of the skeletal organoids to be constructed. Osteoblasts are planted to stimulate the growth of extracellular matrix (ECM). This process is very similar to the growth of human bone tissue and causes the cells to secrete all the proteins needed to complete subsequent functions, ultimately forming a structure that matches the shape of the bone defect area. of skeletal organoids.

Description of drawings

Figure 1 is a schematic diagram of the module structure of the skeletal organoid construction system of the present invention;

Figure 2 is an EM image of the osteoblast layer of the skeletal organoid construction system of the present invention;

Figure 3 is a schematic diagram of bone tissue transplantation in one embodiment of the skeletal organoid construction system of the present invention;

Detailed ways

In order to make the purpose, technical solutions and advantages of the present invention more clear, the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described here are only used to explain the present invention and are not intended to limit the present invention. It should be noted that, as long as there is no conflict, the embodiments and features in the embodiments of the present invention can be combined with each other.

The flow chart in the accompanying drawing is only an exemplary process demonstration. It does not mean that the solution of the present invention must include all the contents, operations and steps in the flow chart, nor does it mean that it must be executed in the order shown in the figure. . For example, some operations/steps in the flow chart can be decomposed, some operations/steps can be combined or partially combined, etc., without departing from the gist of the present invention, the execution sequence shown in the flow chart can be based on the actual situation. Change.

The block diagrams in the accompanying drawings generally represent functional entities and do not necessarily correspond to physically independent entities. That is, these functional entities may be implemented in the form of software or hardware, or one or more operating steps or methods may be used to represent these functional entities, or these functional entities may be implemented in different processing units, operating methods, or experimental steps.

A skeletal organoid construction system of the present invention includes the following modules:

The bone defect data module 1 is used to obtain the volumetric examination data of the patient's bone defect area obtained through medical imaging technology, that is, to obtain the medical imaging data of the patient's bone defect area; the medical imaging data includes X-ray imaging, ultrasound imaging, One or more data from electronic computed tomography (CT), magnetic resonance imaging (MRI), positron emission computed tomography (PET-CT), etc.;

The three-dimensional simulation module 2 is used to convert the acquired medical image data into a three-dimensional image and conduct a three-dimensional simulation of the patient's bone defect area; that is, collect the image data of the patient's bone defect area and restore the original three-dimensional image and defect of the bone defect area. Compare the three-dimensional images of the defective part corresponding to the area, and generate a three-dimensional data model of the skeletal organoid to be constructed based on the three-dimensional image of the bone defect area;

The three-dimensional building module 3 selects an appropriate biocompatible scaffold based on the three-dimensional data model of the skeletal organoid to be constructed, and plants the cultured osteoblast layer on the biocompatible scaffold to allow it to function in the bone microenvironment. During growth, over time, the osteoblast layer grows and forms skeletal organoids.

The three-dimensional building module 3 also includes a microenvironment unit 301, a planting unit 302 and a building unit 303. The microenvironment unit 301 is used to create a microenvironment for the growth of osteoblasts. The microenvironment unit 301 creates an osteogenic microenvironment. The cells include cells related to bone tissue formation, that is, including at least one cell or a combination of cells such as bone marrow stromal cells, osteoprogenitor cells, preosteoblasts, osteoblasts, bone lining cells, osteocytes or osteoclasts. .

In this unit, osteoblast layer and three-dimensional bone tissue can also be cultured at the same time. Cultivating the osteoblast layer means culturing mesenchymal stem cells in a two-dimensional plane and differentiating into the osteoblast layer; culturing three-dimensional bone tissue means Mesenchymal stem cells are three-dimensionally cultured and differentiated into three-dimensional spherical bone tissue.

It should be understood that the osteoblast layer refers to a variety of osteoblasts obtained through differentiation of human stem cells, and further includes osteoblasts cultured from bone tissue. The stem cells mainly refer to mesenchymal stem cells, embryonic stem cells, and embryonic stem cells. Stem cells, pluripotent stem cells and undifferentiated progenitor cells are cells with multi-directional differentiation and renewal, among which mesenchymal stem cells are preferably used.

In the planting unit 302, the cultured osteoblast layer can be planted on a biocompatible scaffold. Specifically, in order to allow the cultured osteoblasts and bone tissue to form a three-dimensional shape closer to that in the human body, a bio-based implant is used. Biocompatible scaffolds made of compatible polymers. This scaffold usually has a groove or mesh structure on which a layer of osteoblasts can be planted. The basic shape or pattern of the biocompatible scaffold promotes the growth of osteoblasts. Osteocytes align or stretch in specific directions, allowing them to grow in an osteogenic microenvironment. This is because biocompatible scaffolds are biodegradable and biocompatible, which means they are compatible with biological systems and degrade naturally within the system without any toxic effects.

It should be known that normal human bone tissue has two different structures: cancellous bone and cortical bone. Cancellous bone is a porous structure with a porosity of 45% to 90%; cortical bone is denser and distributed in the backbone. And the surface of bone tissue has a porosity of 5%-20%. However, regardless of cancellous bone or cortical bone, in order to promote the continuous ingrowth of bone tissue, the interconnected pore structure is very important. This is because the interconnected pores can allow nutrients and oxygen to be transported into the interior of the biocompatible scaffold, promote the growth of cells and bone tissue into the internal structure of the scaffold, promote the formation of vascularization of the scaffold, and remove metabolic products. Therefore, the structure of the above-mentioned biocompatible scaffold can be adjusted according to the actual detected bone defect area to facilitate incubation of three-dimensional bone tissue on the osteoblast layer and induce self-assembly.

The construction unit 303 is used to continuously construct skeletal organoids that are adapted to the bone defect area. According to the different structures and microenvironments of different skeletal organoids, mechanical force is used to simulate the pressure required for human bone formation to convert bone marrow stem cells into A variety of osteoblasts, such as osteoblasts and growth-regulatory osteocytes, required for the growth of the skeletal organoids to be constructed, stimulate the growth of extracellular matrix (ECM). This process is very similar to the growth of human bone tissue and makes the cells Secretes all proteins required to complete subsequent functions. This is because in order for bones to reach their normal shape, they must withstand certain mechanical stresses as they grow. Every time you move, your body weight and muscles put pressure on the bones, which produces minimal deformation. These deformations are necessary for bones to develop to proper size, shape, and strength. Over time, the osteoblasts grow together to form bone tissue and further form bone organoids that match the shape and structure of the bone defect area.

The construction of skeletal organoids of the present invention is suitable for various bones such as femur, tibia, humerus, ulna, radius, meniscus, parietal bone, etc. The corresponding skeletal organoids can be customized according to the needs of the actual bone defect area.

The present invention also relates to a method of using a skeletal organoid construction system, which includes the following steps;

S1: Detect the patient's bone defect area and obtain the patient's volumetric examination data of the bone defect area obtained through medical imaging technology, including X-ray imaging, ultrasound imaging, computed tomography CT, magnetic resonance imaging MRI, and positron emission computer A variety of tomographic imaging PET-CT and other data;

S2: Use computer 3D auxiliary software to convert the acquired medical imaging data into 3D images, perform 3D simulation of the patient's bone defect area, and generate a 3D data model of the skeletal organoids to be constructed;

S3: Select an appropriate biocompatible scaffold based on the three-dimensional data model of the skeletal organoid to be constructed, and plant the cultured osteoblast layer on the biocompatible scaffold to allow it to grow in the bone microenvironment. Over time, the osteoblast layer grows and forms skeletal organoids.

In the step S3, step S301 is also included, which is to use human stem cells to create a microenvironment for the growth of osteoblasts, that is, to create cells related to bone tissue formation, that is, including bone marrow stromal cells, osteoprogenitor cells, preblasts, etc. At least one type of cell or a combination of multiple cells such as osteocytes, osteoblasts, bone lining cells, osteocytes or osteoclasts. The osteoblast layer and the three-dimensional bone tissue can also be cultured at the same time. The osteoblast layer culture means that the mesenchymal stem cells are cultured in a two-dimensional plane and differentiated into the osteoblast layer. The three-dimensional bone tissue culture means that the mesenchymal stem cells are cultured in a two-dimensional plane. Three-dimensional culture and differentiation into three-dimensional spherical bone tissue.

In step S3, step S302 is also included, in which the cultured osteoblast layer is planted on a biocompatible scaffold to form a construct. When using a biocompatible scaffold made of biocompatible polymers, it should be noted that What is unique is that the structure of the biocompatible scaffold is the same or similar to the structure of the bone defect area, and a layer of osteoblasts can be planted on it. The basic shape or pattern of the biocompatible scaffold encourages the growing osteoblasts to grow in a specific direction. Align or stretch.

In step S3, step S303 is also included. In this step, skeletal organoids that are adapted to the bone defect area are continuously constructed. According to the different structures and microenvironments of different skeletal organoids, mechanical force is used to simulate the formation of human bone. The required pressure converts bone marrow stem cells into various osteoblasts such as osteoblasts and growth-regulatory osteocytes required for the growth of the skeletal organoids to be constructed, stimulating the growth of extracellular matrix (ECM) and enabling cell secretion to complete subsequent All proteins required for function. Over time, the osteoblasts grow together to form bone tissue and further form bone organoids that match the shape and structure of the bone defect area.

In one embodiment, a three-dimensional data model and a biocompatible scaffold of the skeletal organoids to be constructed are first generated based on the three-dimensional data of the patient's bone defect area. Mesenchymal stem cells are extracted using existing technology and cultured and proliferated. After that, the Mesenchymal stem cells are seeded on a flat surface and cultured in a 5%-7% CO2 incubator at 36-37.5°C. The cells are treated with osteogenic differentiation medium every 2-4 days, and then replaced with new ones. The culture medium is processed again. It should be noted that the mesenchymal stem cells are differentiated into osteoblast layers and the microenvironment required for bone tissue growth is simultaneously cultivated under the same conditions. Osteogenic differentiation medium contains 30 μg/ml-40 μg/ml ascorbic acid 2-phosphate, 80 nM-120 nM dexamethasone, and 10 ng/ml TGF-β1. TFG-β1 transforming growth factor is used to stimulate mesenchymal stem cells and promote osteogenic differentiation of mesenchymal stem cells. The level of GAG matrix formation was then fixed by treating cells in the osteoblast layer with 10% formaldehyde for 30 min.

When cultivating three-dimensional bone tissue cells, put 3×10 mesenchymal stem cells into a 25ml polypropylene tube and centrifuge for 10 minutes, and then place the mesenchymal stem cells in a 5%-7&CO2 incubator with osteogenic differentiation medium. Incubate at 36.5-37°C for 24-48 hours to generate three-dimensional spherical bone tissue.

The obtained three-dimensional spherical bone tissue is loaded on the differentiated osteoblast layer, and then attached to a biocompatible scaffold that matches the bone defect area. At the same time, the biocompatible scaffold, bone tissue and osteoblasts are The mechanical force is exerted on each layer. Depending on the location of the bone defect, the mechanical force is between 0.8MPa-20MPa. Applying mechanical force can make bone tissue grow into skeletal organoids in an incubator. Of course, as shown in Figure 3, the biocompatible scaffold, bone tissue and osteoblast layer can be directly transplanted into the bone defect area, so that It grows and self-assembles naturally. This is because in order for bones to reach their normal shape, they must withstand certain mechanical stresses as they grow. Every time you move, your body weight and muscles put pressure on the bones, which produces minimal deformation. These deformations are necessary for bones to develop to proper size, shape, and strength. Three-dimensional spherical bone tissue can grow and self-assemble on a biocompatible scaffold. The growing osteoblasts and bone tissue align or stretch in specific directions of the scaffold, and further form skeletal organoids that match the shape and structure of the bone defect area.

The preferred specific implementation modes and examples of the present invention have been described in detail above in conjunction with the accompanying drawings. However, the present invention is not limited to the above-mentioned implementation modes and examples. Within the scope of knowledge possessed by those skilled in the art, other modifications may be made without departing from the present invention. Various changes or equivalent substitutions can be made based on the inventive concept. Therefore, the present invention is not limited to the specific embodiments disclosed here. All embodiments falling within the scope of the claims of the present application are protected by the present invention. In the range.

Claims (10)

1. A system for constructing a skeletal organoid, comprising the following modules:

the bone defect data module (1) is used for acquiring volume inspection data of a bone defect area acquired by a patient through a medical radiography technology and acquiring medical image data of the bone defect area of the patient;

the three-dimensional simulation module (2) is used for converting the acquired medical image data into a three-dimensional image and carrying out three-dimensional simulation on a bone defect area of a patient; generating a three-dimensional data model of the bone organoid to be constructed according to the three-dimensional image of the bone defect area;

and the three-dimensional construction module (3) selects a proper biocompatible bracket according to a three-dimensional data model of the bone organoid to be constructed, and the cultured osteoblast layer is planted on the biocompatible bracket so as to grow in a bone microenvironment, and the osteoblast layer grows and forms the bone organoid along with the growth of time.

2. A bone organoid building system according to claim 1, wherein: the medical contrast data comprises one or more of X-ray imaging, ultrasonic imaging, electronic Computed Tomography (CT), magnetic Resonance Imaging (MRI), positron emission tomography (PET-CT) and the like.

3. A bone organoid building system according to claim 1, wherein: in the process of converting the acquired medical image data into the three-dimensional image, the original three-dimensional image of the bone defect area is reduced and compared with the three-dimensional image of the defect part corresponding to the defect area.

4. A bone organoid building system according to claim 1, wherein: the three-dimensional construction module (3) further comprises a microenvironment unit (301) for providing a growth environment for the osteoblast layer, a planting unit (302) for planting the osteoblast layer on the biocompatible scaffold, and a construction unit (303) for continuously constructing a bone organoid adapted to the bone defect area.

5. The bone organoid building system according to claim 4, wherein: the microenvironment unit (301) is used for creating a microenvironment for osteoblast growth, comprising providing cells involved in bone tissue formation and an environment required for growth of a skeletal organoid to be constructed, while simultaneously culturing osteoblast layers and three-dimensional bone tissue.

6. The bone organoid building system according to claim 5, wherein: the cells related to bone tissue formation comprise at least one cell or a combination of cells such as bone marrow stromal cells, bone progenitor cells, preosteoblasts, osteoblasts, bone lining cells, bone cells or osteoclasts.

7. The bone organoid building system according to claim 4, wherein: the structure of the biocompatible scaffold is determined according to the actually detected bone defect area in the planting unit (302), and the cultured osteoblast layer is planted on the biocompatible scaffold, so that the growing osteoblast is aligned or stretched in a specific direction, and the osteoblast is grown in an osteogenic microenvironment.

8. The bone organoid building system according to claim 4, wherein: the construction unit (303) is used for continuously constructing a bone organoid matched with a bone defect area, simulating the pressure required by human bone formation by using mechanical force according to different bone organoids and different microenvironments, converting bone marrow stem cells into various osteoblasts such as osteoblasts and growth regulating bone cells required by the growth of the bone organoid to be constructed, and further forming the bone organoid matched with the shape and structure of the bone defect area.

9. A method of using a system for constructing a skeletal organoid, comprising the steps of:

s1: detecting a bone defect area of a patient, and acquiring volume inspection data of the bone defect area, which is acquired by the patient through a medical radiography technology;

s2: converting the acquired medical image data into a three-dimensional image by using computer three-dimensional auxiliary software, and performing three-dimensional simulation on a bone defect area of a patient to generate a three-dimensional data model of a bone organoid to be constructed;

s3: according to the three-dimensional data model of the bone organoid to be constructed, a proper biocompatible scaffold is selected, and a cultured osteoblast layer is planted on the biocompatible scaffold, so that the osteoblast layer grows in a bone microenvironment, and grows and forms the bone organoid along with the time.

10. The method of using a bone organoid building system according to claim 9, wherein; in the step S3, the method further includes a step S301 of creating a microenvironment for osteoblast growth, that is, creating cells related to bone tissue formation, by using the human stem cells, and simultaneously culturing osteoblast layers and three-dimensional bone tissue;

further comprising step S302, planting the cultured osteoblast layer on a biocompatible scaffold adapted to the bone defect area to form a construct;

and step S303, continuously constructing a bone organoid matched with the bone defect area, and simulating the pressure required by human bone formation by using mechanical force according to different structures and microenvironments of different bone organoids so as to convert bone marrow stem cells into various osteoblasts such as osteoblasts, growth regulating bone cells and the like required by the growth of the bone organoid to be constructed.