Method for constructing injectable bone organ to realize rapid bone regeneration

Abstract

The invention relates to a method for constructing an injectable bone organ to realize rapid bone regeneration. The method comprises the following steps: 1, obtaining a water-soluble periosteum acellular matrix by adopting an acellular technology; secondly, compound bone salt is prepared according to the proportion of natural bone salt components, the water-soluble periosteum acellular matrix and the compound bone salt are mixed according to the proportion of natural bone organic and inorganic components to form a bionic matrix component, and then the bionic matrix component is added into gelatin to construct a completely bionic osteogenesis microenvironment; and 3, constructing a bone regeneration material to form a bone organ, firstly preparing a granular gelatin scaffold with a full-bionic osteogenesis microenvironment, then inoculating the bone marrow mesenchymal stem cells, finally performing in-vitro culture, and regulating the differentiation and secretion of the bone marrow mesenchymal stem cells by virtue of the osteogenesis microenvironment of the scaffold to finally form the bone organ.

Description

Technical Field

[0001] The present invention relates to the field of biological bone regeneration cells, in particular to a method for constructing injectable bone organoids to achieve rapid bone regeneration. Background Art

[0002] Bone defects are common in clinical practice and are often difficult to regenerate naturally. However, many refractory bone defects require human intervention. The emergence of tissue engineering offers an ideal therapeutic strategy, combining scaffold materials, microenvironmental structures, and seed cells to synergistically repair bone defects. However, the complexity of most bone defects, coupled with the clinical focus on minimally invasive repair, necessitates a repair strategy with a strong osteogenesis effect and ease of clinical application.

[0003] International Publication No. WO2011 / 108537 describes a cell support composed of a porous body, which is useful as a bone regeneration material, but its cartilage regeneration performance is unclear. The porous body is composed of a biodegradable material and has a specified porosity, a specified average pore size, interconnected pores between pores, and a specified water absorption rate. Japanese Patent Application Publication No. 2001-519210 describes a multilayer membrane comprising a matrix layer primarily comprising type II collagen and having a sponge-like open structure, and at least one barrier layer having a relatively impermeable closed structure. Patent Document 2 describes this multilayer membrane as being particularly suitable for in vivo regeneration of bone tissue or cartilage tissue.

[0004] There are currently no reports on screening out which concentration of granular gelatin is suitable for bone regeneration, and there are currently no reports on injectable organoids with a fully biomimetic combination of inorganic mineralization microenvironment and organic osteogenic microenvironment to achieve rapid bone regeneration.

[0005] This invention focuses on clinical practice, with the goal of injectable repair, to construct injectable organoids with a strong mineralized microenvironment and an organic bionic microenvironment to achieve bone regeneration. Summary of the Invention

[0006] The main purpose of the present invention is to overcome the above-mentioned shortcomings of the prior art and to provide a method for constructing injectable bone organoids to achieve rapid bone regeneration.

[0007] The present invention is achieved by the following technical solutions:

[0008] A method for constructing injectable bone organoids to achieve rapid bone regeneration is characterized by: in the first step, a water-soluble periosteum decellularized matrix is ​​obtained using decellularization technology; in the second step, a compound bone salt is prepared according to the ratio of natural bone salt components, and the water-soluble periosteum decellularized matrix and the compound bone salt are mixed according to the ratio of natural bone organic and inorganic components to form a biomimetic matrix component, and then the biomimetic matrix component is added to gelatin to construct a completely biomimetic osteogenic microenvironment; in the third step, bone regeneration materials are constructed to form bone organoids, first preparing a granular gelatin scaffold with a completely biomimetic osteogenic microenvironment → then inoculating bone marrow mesenchymal stem cells → finally culturing in vitro, relying on the osteogenic microenvironment of the scaffold to regulate the differentiation and secretion of bone marrow mesenchymal stem cells to ultimately form bone organoids.

[0009] Further, the first step of obtaining a water-soluble periosteum decellularized matrix using a decellularization technique is as follows:

[0010] The porcine tibia was harvested and the periosteum was harvested. The periosteum was then subjected to ice crystal formation and fragmentation (the periosteum was placed in liquid nitrogen for 3-5 minutes, then immediately placed in a 37° shaker to thaw, and this process was repeated five times to allow intracellular ice crystals to form and pierce the cell membrane, facilitating subsequent decellularization). The membrane was then broken with a 5% Triton-X100 solution for 48 hours, with the solution changed every 12 hours. The membrane was then treated with 10mM Tris HCl for 48 hours, with the solution changed every 12 hours. Residual nucleic acids were then removed using 100U / ml DNase and 5U / ml RNase. The matrix was then digested with 1mg / ml pepsin at pH 2, adjusted to pH 7, and lyophilized to complete the preparation of a water-soluble acellular matrix.

[0011] Further described, the second step of constructing a completely biomimetic osteogenic microenvironment is as follows:

[0012] The compound bone salt is prepared according to the proportion of natural bone salt components, and the water-soluble periosteum decellularized matrix and the compound bone salt are mixed according to the ratio of natural bone organic and inorganic components (organic: inorganic = 3:7) to form a bionic matrix component. The bionic matrix component is then added to gelatin to construct a completely bionic osteogenic microenvironment.

[0013] Preferably, the compound bone salt composition is composed of a mixture of 84% calcium phosphate, 10% calcium carbonate, 2% calcium citrate, 2% magnesium phosphate, and 2% disodium hydrogen phosphate.

[0014] Furthermore, the third step of constructing the bone regeneration material is as follows:

[0015] (5) 0.4%, 0.6%, 0.8%, and 1% gelatin were dissolved in deionized water, and then placed in -4° for 12 h, -10° for 12 h, -20° for 8 h, and -80° for 4 h in a gradient and steady cooling process to obtain a scaffold with uniform pore size.

[0016] The goal is to ensure uniform ice crystal formation, resulting in a scaffold with uniform pore size, which is more conducive to homogeneous tissue regeneration. At this point, the scaffold is not yet complete. It still needs to be freeze-dried in a freeze dryer, cross-linked after freeze-drying (otherwise, it will melt in water). After cross-linking, the excess cross-linker is washed off and freeze-dried again to produce the final gelatin scaffold.

[0017] (6) freeze-drying the uniform pore size scaffold;

[0018] Preferably, the freeze-drying process is to freeze-dry the gelatin scaffold in a freeze dryer for 24 hours.

[0019] The purpose is to sublime ice crystals and prepare the porous structure of the scaffold.

[0020] (7) Dissolve the crosslinking agents 0.5% EDC and 0.3% NHS in 95% ethanol, use this solution to crosslink the freeze-dried gelatin scaffold for 12 h, then wash away the excess crosslinker with deionized water and freeze-dry again; use scissors to cut the freeze-dried gelatin into small pieces of 2x2 mm for later use.

[0021] Specifically, EDC (dimethylaminopropyl) carbodiimide; NHS (N-hydroxysuccinimide). The crosslinker is dissolved in 95% ethanol and allowed to soak in the freeze-dried gelatin scaffold for 12 hours at room temperature to complete crosslinking. The scaffold is cut into small pieces for injectability.

[0022] (8) The above granular gelatin scaffold was subjected to cell suspension (1x10 7 The scaffolds were inoculated with 5% 4% 5-8% 1% 4% 2-6% 1% 1% 2-antibody (CSH) and 4% 2-6% 1% 2-antibody (CSH). After incubation for 2-4 hours, the culture medium was added and the scaffolds were cultured in low-glucose DMEM containing 10% fetal bovine serum and 1% double-antibody for 7 days. After that, the scaffolds were centrifuged and loaded into syringes for rapid bone regeneration.

[0023] Beneficial effects of the present invention: Due to the adoption of the above technical solution, the present invention focuses on clinical practice, takes injectable repair as the goal, constructs injectable organoids with a strong mineralized microenvironment and an organic bionic microenvironment, and realizes bone regeneration.

[0024] The present invention will be further described below with reference to the accompanying drawings and embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 is a schematic diagram of the preparation and characterization of the embodiment of the present invention;

[0026] Figure 2 This is a schematic diagram showing the best mineralization achieved by 0.8% gelatin in an embodiment of the present invention;

[0027] Figure 3 Schematic diagram of the preparation and characterization of the inorganic-organic dual microenvironment gelatin scaffold according to an embodiment of the present invention;

[0028] Figure 4 This is a schematic diagram of an embodiment of the present invention showing that a dual microenvironment biomimetic osteogenic scaffold can cooperate with bone marrow mesenchymal stem cells to achieve good mineralization;

[0029] Figure 5 This is a schematic diagram of an embodiment of the present invention showing that a dual microenvironment biomimetic osteogenic scaffold can cooperate with bone marrow mesenchymal stem cells to achieve good organic reconstruction;

[0030] Figure 6 is an injectable schematic diagram of an embodiment of the present invention;

[0031] Figure 7 This is a schematic diagram showing that the scaffold of the present invention has good biocompatibility and can induce bone marrow mesenchymal stem cells to secrete bone-specific proteins, indicating the successful construction of organoids;

[0032] Figure 8 This is a schematic diagram of the construction, identification, and in vivo regeneration of bone organoids according to an embodiment of the present invention. DETAILED DESCRIPTION

[0033] The following is a clear and complete description of the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0034] like Figure 1-6 The illustrated embodiment provides a method for constructing injectable bone organoids to achieve rapid bone regeneration. The first step is to obtain a water-soluble periosteum decellularized matrix using decellularization technology. The second step is to prepare a compound bone salt based on the ratio of natural bone salt components, and mix the water-soluble periosteum decellularized matrix and the compound bone salt according to the ratio of natural bone organic and inorganic components to form a biomimetic matrix component. The biomimetic matrix component is then added to gelatin to construct a completely biomimetic osteogenic microenvironment. The third step is to construct a bone regeneration material to form a bone organoid. First, a granular gelatin scaffold with a completely biomimetic osteogenic microenvironment is prepared, then bone marrow mesenchymal stem cells are inoculated, and finally, in vitro culture is performed. The osteogenic microenvironment of the scaffold is relied upon to regulate the differentiation and secretion of the bone marrow mesenchymal stem cells to ultimately form the bone organoid.

[0035] In the specific implementation process, the steps of obtaining water-soluble periosteum decellularized matrix using decellularization technology are as follows:

[0036] The porcine tibia was harvested and the periosteum was harvested. The periosteum was then subjected to ice crystal formation and fragmentation (the periosteum was placed in liquid nitrogen for 3-5 minutes, then immediately placed in a 37° shaker to thaw, and this process was repeated five times to allow intracellular ice crystals to form and pierce the cell membrane, facilitating subsequent decellularization). The membrane was then broken with a 5% Triton-X100 solution for 48 hours, with the solution changed every 12 hours. The membrane was then treated with 10mM Tris HCl for 48 hours, with the solution changed every 12 hours. Residual nucleic acids were then removed using 100U / ml DNase and 5U / ml RNase. The matrix was then digested with 1mg / ml pepsin at pH 2, adjusted to pH 7, and lyophilized to complete the preparation of a water-soluble acellular matrix.

[0037] In the specific implementation process, the second step of constructing a completely bionic osteogenic microenvironment is as follows:

[0038] The compound bone salt is prepared according to the proportion of natural bone salt components, and the water-soluble periosteum decellularized matrix and the compound bone salt are mixed according to the ratio of natural bone organic and inorganic components (organic: inorganic = 3:7) to form a bionic matrix component. The bionic matrix component is then added to gelatin to construct a completely bionic osteogenic microenvironment.

[0039] In the specific implementation process, the compound bone salt is composed of a mixture of 84% calcium phosphate, 10% calcium carbonate, 2% calcium citrate, 2% magnesium phosphate, and 2% disodium hydrogen phosphate.

[0040] During the specific implementation process, the third step of constructing bone regeneration materials is as follows:

[0041] 0.4%, 0.6%, 0.8% and 1% gelatin were dissolved in deionized water, and then placed in -4° for 12h, -10° for 12h, -20° for 8h and -80° for 4h in sequence to form pores by gradient cooling to obtain a scaffold with uniform pore size.

[0042] The goal is to ensure uniform ice crystal formation, resulting in a scaffold with uniform pore size, which is more conducive to homogeneous tissue regeneration. At this point, the scaffold is not yet complete. It still needs to be freeze-dried in a freeze dryer, cross-linked after freeze-drying (otherwise, it will melt in water). After cross-linking, the excess cross-linker is washed off and freeze-dried again to produce the final gelatin scaffold.

[0043] In a specific implementation process, the uniform pore size scaffold is freeze-dried;

[0044] In a specific implementation process, the freeze-drying process is to freeze-dry the gelatin scaffold in a freeze dryer for 24 hours.

[0045] The purpose is to sublime ice crystals and prepare the porous structure of the scaffold.

[0046] In the specific implementation process, the crosslinking agents 0.5% EDC and 0.3% NHS were dissolved in 95% ethanol, and the freeze-dried gelatin scaffold was cross-linked with the solution for 12 hours. The excess crosslinker was then washed with deionized water and freeze-dried again. The freeze-dried gelatin was cut into 2x2mm small pieces with scissors for later use.

[0047] Specifically, EDC (dimethylaminopropyl) carbodiimide; NHS (N-hydroxysuccinimide). The crosslinker is dissolved in 95% ethanol and allowed to soak in the freeze-dried gelatin scaffold for 12 hours at room temperature to complete crosslinking. The scaffold is cut into small pieces for injectability.

[0048] In the specific implementation process, the above-mentioned granular gelatin scaffold was subjected to cell suspension (1×10 7 The scaffold was inoculated with 1% 400 μg / ml of culture medium and incubated for 2-4 h before adding culture medium. The scaffold was then cultured in low-glucose DMEM containing 10% fetal bovine serum and 1% double antibody for 7 days, and then centrifuged. Figure 6 The syringe shown is used to achieve rapid bone regeneration.

[0049] The design principle of this project is as follows: Bone marrow mesenchymal stem cells were seeded with four concentrations of granular gelatin and then transplanted subcutaneously into nude mice to achieve bone regeneration. Based on the quality of the regenerated bone, 0.8% gelatin was determined to be most beneficial for bone regeneration. This was the point at which the gelatin concentration was selected. Subsequently, because gelatin lacks an osteogenic microenvironment, we sought to enhance it from both inorganic and organic perspectives. The organic component used was periosteum decellularized matrix, as extensive evidence suggests that periosteum is a potent osteogenic organic component. Furthermore, natural bone inorganic salts also have a strong osteogenic effect. We constructed an inorganic osteogenic microenvironment modeled on the composition and ratio of inorganic salts found in natural bone (84% calcium phosphate, 10% calcium carbonate, 2% calcium citrate, 2% magnesium phosphate, and 2% disodium hydrogen phosphate). Since the organic:inorganic ratio in natural bone is 3:7, we chose to create a mixed additive consisting of periosteum decellularized matrix and formulated inorganic salts at a ratio of 3:7, mimicking that of natural bone. However, it is not clear how much mixed additives can maintain the 0.8% gelatin characterization without significant changes. We prepared gelatin scaffolds with 10%, 30%, and 50% mixed additives and found that 30% did not significantly affect the characterization, while 50% did. Therefore, a gelatin particle scaffold with 30% mixed additives was selected to prepare subsequent bone organoids. The selected scaffold was prepared into granules and loaded with bone marrow mesenchymal stem cells. It was found that it can effectively promote the expression of bone-specific genes and proteins and activate signaling pathways related to osteogenesis and extracellular matrix deposition. We transplanted bone organoids into the rabbit's subcutaneous tissue and found that they can achieve good bone regeneration in 4 weeks, while most osteogenic constructs require 8-12 weeks to achieve good bone regeneration. Thus, a method for constructing injectable bone organoids to achieve rapid bone regeneration was constructed.

[0050] The above descriptions are merely embodiments of the present invention and are not intended to limit the patent scope of the present invention. Any equivalent structure or equivalent process transformation made using the contents of the present invention specification, or directly or indirectly applied in other related technical fields, are also included in the patent protection scope of the present invention.

Claims

1. A method for constructing injectable bone organoids to achieve rapid bone regeneration, characterized in that: The first step is to use decellularization technology to obtain water-soluble periosteum decellularized matrix; the second step is to prepare compound bone salt according to the proportion of natural bone salt components, and mix the water-soluble periosteum decellularized matrix and compound bone salt according to the proportion of natural bone organic and inorganic components to form a bionic matrix component, and then add the bionic matrix component to gelatin to construct a completely bionic osteogenic microenvironment; the third step is to construct bone regeneration materials to form bone organoids, first prepare a granular gelatin scaffold with a fully bionic osteogenic microenvironment → then inoculate bone marrow mesenchymal stem cells → finally culture in vitro, relying on the osteogenic microenvironment of the scaffold to regulate the differentiation and secretion of bone marrow mesenchymal stem cells to finally form bone organoids.

2. A method for constructing injectable bone organoids to achieve rapid bone regeneration according to claim 1, characterized in that: The first step of using decellularization technology to obtain water-soluble periosteum decellularized matrix is ​​as follows: The pig's tibia was taken and the tibia periosteum was obtained, and then ice crystal formation and crushing were performed in sequence (the periosteum was placed in liquid nitrogen for 3-5 minutes, and then immediately placed in a 37° shaker to melt, and this operation was repeated 5 times, the purpose was to allow ice crystals to form in the cells to pierce the cell membrane, which was convenient for subsequent decellularization), 5% Triton-X100 solution was used for membrane breaking for 48 hours, and the solution was changed every 12 hours, and 10mM Tris HCl was used for 48 hours, and the solution was changed every 12 hours, and then 100U / ml DNase and 5U / ml RNase were used to remove residual nucleic acids. Then, 1mg / ml pepsin was used for digestion in a liquid with pH = 2, and then the pH was adjusted to 7 and freeze-dried to complete the preparation of water-soluble decellularized matrix.

3. A method for constructing injectable bone organoids to achieve rapid bone regeneration according to claim 1, characterized in that: The second step to construct a completely bionic osteogenic microenvironment is as follows: The compound bone salt is prepared according to the proportion of natural bone salt components, and the water-soluble periosteum decellularized matrix and the compound bone salt are mixed according to the proportion of natural bone organic and inorganic components to form a bionic matrix component, and then the bionic matrix component is added to gelatin to construct a completely bionic osteogenic microenvironment.

4. A method for constructing injectable bone organoids to achieve rapid bone regeneration according to claim 3, characterized in that: Organic:inorganic=3:

7.

5. A method for constructing injectable bone organoids to achieve rapid bone regeneration according to claim 1, characterized in that: The compound bone salt is composed of a mixture of 84% calcium phosphate, 10% calcium carbonate, 2% calcium citrate, 2% magnesium phosphate, and 2% disodium hydrogen phosphate.

6. A method for constructing injectable bone organoids to achieve rapid bone regeneration according to claim 1, characterized in that: The third step is to construct bone regeneration materials. The specific steps are as follows: (1) Dissolve 0.4%, 0.6%, 0.8%, and 1% gelatin in deionized water, and then place them in -4° for 12 h, -10° for 12 h, -20° for 8 h, and -80° for 4 h to form pores in a gradient and steady cooling manner to obtain a scaffold with uniform pore size; (2) freeze-drying the uniform pore size scaffold; Preferably, the freeze-drying process is to freeze-dry the gelatin scaffold in a freeze dryer for 24 hours; (3) Dissolve the crosslinking agents 0.5% EDC and 0.3% NHS in 95% ethanol, use the solution to crosslink the freeze-dried gelatin scaffold for 12 h, then wash away the excess crosslinking agent with deionized water and freeze-dry again; use scissors to cut the freeze-dried gelatin into small pieces of 2x2 mm for later use; (4) The above-mentioned granular gelatin scaffold was subjected to cell suspension (1x10 7 The scaffolds were inoculated with 1% 5-6-nitropropene / ml of cellulose, incubated for 2-4 hours, and then the culture medium was added. The scaffolds were then cultured in low-glucose DMEM containing 10% fetal bovine serum and 1% double antibody for 7 days. After that, they were centrifuged and loaded into syringes for rapid bone regeneration.

7. A method for constructing injectable bone organoids to achieve rapid bone regeneration according to claim 6, characterized in that: Specifically, EDC: (dimethylamino)propyl) carbodiimide; NHS N-hydroxysuccinimide; the scaffold is dissolved with 95% ethanol and the cross-linking agent is soaked in the first freeze-dried gelatin scaffold at room temperature for 12 hours to complete the cross-linking; the scaffold is cut into small pieces for injectability.

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