Method for promoting diabetic fracture healing by using bone marrow mesenchymal stem cell exosome

Abstract

The invention discloses a method for promoting diabetic fracture healing by using bone marrow mesenchymal stem cell exosomes, which comprises the following steps: separating MSCs (mesenchymal stem cells) from the bone marrow of a diabetic patient, culturing and extracting the exosomes; the method comprises the following steps: extracting an exosome from a bone fracture part, performing quantitative analysis on the extracted exosome, injecting the exosome into the bone fracture part in a manner of intravenous injection, local injection or minimally invasive injection, continuously treating, promoting proliferation and differentiation of osteoblasts by miRNA or mRNA in the exosome through targeting cells, enhancing angiogenesis, and further accelerating fracture healing. According to the exosome, proliferation and differentiation of osteoblasts are promoted through miRNA or mRNA in the exosome, the synthesis capacity of bone matrixes of the osteoblasts is enhanced, so that fracture healing is accelerated, meanwhile, the exosome provides necessary blood supply for fracture healing by adjusting vascular endothelial cells and promoting angiogenesis, and the repair capacity of fracture parts is effectively improved.

Description

Technical Field

[0001] The present invention relates to the technical field of fracture repair, and in particular to a method for promoting diabetic fracture healing by using bone marrow mesenchymal stem cell exosomes. Background Art

[0002] Diabetes is a widespread metabolic disease, and patients often face delayed or incomplete fracture healing. Diabetes affects bone metabolism through a variety of mechanisms, including bone cell dysfunction, bone loss, and decreased bone density caused by high blood sugar. These factors lead to a significant decrease in the fracture healing ability of diabetic patients. Existing treatments for diabetic fracture healing mostly focus on controlling blood sugar levels and using drugs to promote bone repair, but the effects of these methods are not ideal and may be accompanied by certain side effects;

[0003] Existing drug intervention and surgical repair treatment methods have alleviated the fracture healing problem in diabetic patients to a certain extent. However, due to the particularity of diabetes, it is often impossible to effectively restore the normal process of fracture healing. In particular, traditional treatment methods fail to target the bone healing disorders caused by insufficient bone formation and blocked angiogenesis caused by diabetes. Based on this, a method of promoting diabetic fracture healing with bone marrow mesenchymal stem cell exosomes is needed. Summary of the invention

[0004] To achieve the above object, the present invention provides the following scheme: a method for promoting diabetic fracture healing by bone marrow mesenchymal stem cell exosomes, comprising the following steps:

[0005] Step 1: Bone marrow mesenchymal stem cells are isolated from the bone marrow of diabetic patients, and exosomes secreted by the stem cells are extracted by culture. The exosomes are separated by ultra-high speed centrifugation at a speed of 100,000 rpm for 3 hours. The exosomes are purified by dialysis or immunoaffinity method;

[0006] Step 2: Quantitative analysis of the extracted exosomes, the concentration of the exosomes is 10^9 to 10^12 particles / ml, preferably 10^10 particles / ml;

[0007] Step 3: injecting the exosomes into the fracture site of the diabetic patient by intravenous injection, local injection or minimally invasive injection, with an injection dose of 10^10 particles / ml, once a week, and continuing the treatment for 4 to 6 weeks;

[0008] Step 4: The exosome dose for each injection is 10^10 particles / ml, once a week, and the treatment lasts for 4-6 weeks. The specific treatment cycle is adjusted appropriately according to the patient's healing progress;

[0009] Step 5: Evaluate the progress of fracture healing in diabetic patients through imaging examinations and bone density measurements, and adjust the treatment plan based on the examination results.

[0010] Further preferably, the exosomes are extracted by ultra-high-speed centrifugation and purified by dialysis or immunoaffinity to a purity of more than 85%, thereby ensuring that the exosomes are not interfered by cell debris and other contaminants during the extraction process;

[0011] The extraction method of exosomes from bone marrow mesenchymal stem cells explicitly uses ultra-high-speed centrifugation, a common and efficient method for isolating exosomes. Ultra-high-speed centrifugation can separate exosomes from cell culture supernatant because the size and density of exosomes enable them to be effectively distinguished from other larger or smaller particles under high-speed centrifugation. The extracted exosomes also need to be purified to ensure their quality and function. The exosomes are purified by using dialysis or immunoaffinity. The dialysis method removes small molecule impurities from the exosome solution through a semi-permeable membrane. The immunoaffinity method helps remove non-specific substances by using specific antibodies to capture specific markers on the surface of exosomes, ensuring that the extracted exosomes have a high purity. The purification methods together ensure that the extracted exosomes have a high purity, usually reaching more than 85%.

[0012] Further preferably, the exosomes contain one of miRNA or mRNA, the miRNA includes miR-21 and miR-29b, the miRNA or mRNA enters the target cells through the transport of exosomes, the miRNA or mRNA is used to intervene in the cell behavior at the fracture site, and the selection of the miRNA and mRNA is based on their role in bone and soft tissue repair; by selecting specific miRNA or mRNA, it is transported to the target cells at the fracture site through exosomes, thereby regulating the gene expression of the cells and promoting fracture healing. Exosomes serve as natural delivery carriers to effectively protect miRNA or mRNA, ensuring that it crosses the cell membrane and enters the target cells. After entering the target cells, miRNA or mRNA regulates cell proliferation, differentiation and function by activating related genes of osteoblasts, chondrocytes and vascular endothelial cells, thereby promoting the repair and regeneration of bones and soft tissues.

[0013] Further preferably, the concentration range of the exosomes is 10^9 to 10^12 particles / ml, and the concentration of the exosomes is adjusted according to the clinical manifestations and course of the diabetic patient; the concentration range of the exosomes is set between 10^9 and 10^12 particles / ml, which can meet the treatment needs of different patients. On the basis of the clinical manifestations and course of the diabetic patient, the concentration of the exosomes is adjusted according to the severity of the patient's condition or the specific progress of the healing process to improve the accuracy and effect of the treatment. The appropriate concentration can ensure the effective use of exosomes in the treatment process, avoiding the insignificant therapeutic effect due to too low concentration, or the potential side effects caused by too high concentration.

[0014] Further preferably, the exosomes are injected into the fracture site of the diabetic patient by intravenous injection, local injection or minimally invasive injection. The local injection uses a fine needle to precisely inject the exosomes into the fracture area. The minimally invasive injection assists the delivery of exosomes by using a biocompatible carrier.

[0015] In the treatment of diabetic fracture patients, choosing an appropriate injection method can enable exosomes to be directly and effectively delivered to the fracture site and promote fracture healing;

[0016] Intravenous injection: Exosomes are directly delivered into the body through intravenous injection and distributed to the fracture site through systemic circulation. Intravenous injection is suitable for situations where a wider effect or treatment of multiple fracture sites is required;

[0017] Local injection: Using a fine needle to precisely inject exosomes into the fracture area can ensure that the exosomes act directly on the target site, reduce systemic side effects, and ensure a higher local treatment effect. Local injection is the choice for treating a single fracture or a specific area;

[0018] Minimally invasive injection: Use biocompatible carriers to assist the delivery of exosomes, and inject exosomes into the fracture site through minimally invasive technology. Minimally invasive injection can reduce the trauma of patients while maintaining the high efficiency of treatment. It is suitable for patients who require less intervention and a shorter recovery period.

[0019] Further preferably, during the treatment process, dynamic imaging examinations combined with bone density tests and fracture healing assessment indicators are used to monitor the bone healing status in real time to guide the adjustment of the treatment plan and ensure that the use of exosomes is compatible with the patient's fracture healing process.

[0020] Further preferably, the miRNA or mRNA in the exosomes can interact with the target cells through the cell membrane during the treatment process, activating osteoblasts and vascular endothelial cells related cells;

[0021] The miRNA or mRNA in exosomes interacts with target cells through the cell membrane. After entering the target cells, it can play its regulatory role. The molecules promote the target cells to perform their due biological functions by regulating gene expression, inhibiting or activating specific signaling pathways. Osteoblasts are the key cell type for bone repair and can accelerate fracture healing by secreting bone matrix and promoting mineralization. miRNA or mRNA regulates the gene expression of osteoblasts, activates their differentiation, proliferation and mineralization functions, and enhances the efficiency of fracture healing. In the early stages of fracture healing, angiogenesis is the basis of bone repair. The miRNA or mRNA in exosomes regulates the behavior of vascular endothelial cells and promotes the formation of new blood vessels, thereby providing sufficient oxygen and nutrients for bone healing.

[0022] Further preferably, the carrier of the exosomes is selected to be liposomes, nanoparticles or other biocompatible materials;

[0023] Liposomes are nanoparticles composed of a lipid bilayer membrane. They have good biocompatibility and low toxicity, and can effectively encapsulate and protect the active ingredients in exosomes. The membrane structure of liposomes can help exosomes avoid premature degradation in the body, thereby extending their half-life in the body and enhancing the stability of exosomes. Nanoparticles as carriers can bring exosomes into target cells by directly penetrating the cell membrane or by endocytosis, effectively improving the therapeutic effect. In addition to liposomes and nanoparticles, other biocompatible materials can also serve as carriers of exosomes and can combine with exosomes to form stable complexes, which help to improve the stability, delivery efficiency and targeting of exosomes.

[0024] According to the specific embodiments provided by the present invention, the present invention discloses the following technical effects:

[0025] The exosomes of the present invention are small vesicles secreted by bone marrow mesenchymal stem cells, which can carry a variety of biologically active substances. The active substances can be effectively transported to the target cells at the fracture site through the transport effect of the exosomes, regulate the relevant biological processes of fracture healing, promote the proliferation and differentiation of osteoblasts through the miRNA or mRNA in the exosomes, enhance their ability to synthesize bone matrix, thereby accelerating fracture healing. At the same time, exosomes regulate vascular endothelial cells, promote angiogenesis, provide the necessary blood supply for fracture healing, thereby effectively improving the repair ability of the fracture site. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0027] Figure 1 It is a flow chart of the steps of the present invention. DETAILED DESCRIPTION

[0028] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe 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 creative work are within the scope of protection of the present invention.

[0029] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, the present invention is further described in detail below with reference to the accompanying drawings and specific embodiments.

[0030] Example

[0031] like Figure 1 As shown, a method for promoting diabetic fracture healing by bone marrow mesenchymal stem cell exosomes in this embodiment includes the following steps:

[0032] Step 1: Extraction and purification of exosomes. Bone marrow mesenchymal stem cells (MSCs) are isolated from the bone marrow of diabetic patients, and secreted exosomes are obtained through cell culture. Exosomes are separated by ultra-high-speed centrifugation at a speed of 100,000 rpm for 3 hours. The extracted exosomes are purified by dialysis or immunoaffinity to ensure that the purity of the exosomes reaches more than 85% and avoid interference from cell debris and other contaminants.

[0033] Step 2: Exosome quantification and concentration adjustment: Quantitative analysis of the extracted exosomes was performed to ensure that their concentration was between 10^9 and 10^12 particles / ml. To ensure the therapeutic effect, the preferred concentration was 10^10 particles / ml. The concentration of exosomes was appropriately adjusted according to the clinical manifestations and course of the diabetic patient to meet the needs of individualized treatment.

[0034] Step 3: Exosome injection and treatment plan: exosomes are injected into the fracture site of diabetic patients by intravenous injection, local injection or minimally invasive injection. Local injection uses a fine needle to accurately inject exosomes into the fracture area, while minimally invasive injection uses a biocompatible carrier to assist the delivery of exosomes. The exosome concentration of each injection is 10^10 particles / ml, and the injection is given once a week for 4 to 6 weeks. The specific treatment cycle is adjusted according to the patient's healing progress.

[0035] Step 4: Dynamic imaging monitoring and treatment adjustment. During the treatment process, X-ray, CT or MRI dynamic imaging examinations and bone density tests are used to monitor the fracture healing in real time. Combined with fracture healing evaluation indicators, the fracture healing process is evaluated, and the injection dose and treatment cycle of exosomes are adjusted in time according to the examination results to ensure that the treatment plan is compatible with the patient's healing process.

[0036] Step 5: The role of miRNA and cell intervention. Exosomes contain miRNA, especially miR-21 and miR-29b. MiRNA enters the target cells through exosomes, regulates the behavior of related cells such as osteoblasts and vascular endothelial cells, and promotes fracture healing. MiR-21 promotes the deposition of bone matrix by promoting osteoblast proliferation and differentiation, while miR-29b further promotes bone healing by improving angiogenesis. During the treatment process, the miRNA in the exosomes can activate the signaling pathways of related cells and promote cell repair and bone reconstruction at the fracture site.

[0037] Example 2

[0038] Different from Example 1, a method for promoting diabetic fracture healing by bone marrow mesenchymal stem cell exosomes comprises the following steps:

[0039] Step 1: Extraction and purification of exosomes. Bone marrow mesenchymal stem cells (MSCs) are isolated from the bone marrow of diabetic patients, and secreted exosomes are obtained through cell culture. Exosomes are separated by ultra-high-speed centrifugation at a speed of 100,000 rpm for 3 hours. The extracted exosomes are purified by dialysis or immunoaffinity to ensure that the purity of the exosomes reaches more than 85% and avoid interference from cell debris and other contaminants.

[0040] Step 2: Exosome quantification and concentration adjustment: Quantitative analysis of the extracted exosomes was performed to ensure that their concentration was between 10^9 and 10^12 particles / ml. To ensure the therapeutic effect, the preferred concentration was 10^12 particles / ml. According to the clinical manifestations and course of the diabetic patient, the concentration of exosomes was appropriately adjusted to meet the needs of individualized treatment.

[0041] Step 3: Exosome injection and treatment plan: Inject exosomes into the fracture site of diabetic patients by intravenous injection, local injection or minimally invasive injection; local injection uses a fine needle to precisely inject exosomes into the fracture area, and minimally invasive injection uses a biocompatible carrier to assist the delivery of exosomes; the exosome concentration of each injection is 10^12 particles / ml, and the injection is given once a week for 4 to 6 weeks. The specific treatment cycle is adjusted according to the patient's healing progress;

[0042] Step 4: Dynamic imaging monitoring and treatment adjustment. During the treatment process, dynamic imaging examinations and bone density tests are used to monitor the fracture healing in real time. Combined with fracture healing evaluation indicators, the fracture healing process is evaluated, and the injection dose and treatment cycle of exosomes are adjusted in time according to the examination results to ensure that the treatment plan is compatible with the patient's healing process.

[0043] Step 5: The role of mRNA and cell intervention. Exosomes contain mRNA, especially mRNA related to bone formation. mRNA enters the target cells through exosomes, promotes the proliferation and differentiation of osteoblasts, vascular endothelial cells and other cells, and promotes fracture healing. mRNA can regulate the synthesis of related proteins through gene expression, enhance bone matrix formation and angiogenesis, and accelerate the repair process of the fracture site.

[0044] The technical features of the above embodiments may be combined arbitrarily. To make the description concise, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0045] The principles and implementation methods of the present invention are described in this article using specific examples. The description of the above embodiments is only used to help understand the method and core idea of ​​the present invention. At the same time, for those skilled in the art, according to the idea of ​​the present invention, there will be changes in the specific implementation methods and application scope. In summary, the content of this specification should not be understood as limiting the present invention.

Claims

1. A method for promoting diabetic fracture healing by bone marrow mesenchymal stem cell exosomes, characterized in that: The following steps are involved: Step 1: Bone marrow mesenchymal stem cells are isolated from the bone marrow of diabetic patients, and exosomes secreted by the stem cells are extracted by culture. The exosomes are separated by ultra-high speed centrifugation at a speed of 100,000 rpm for 3 hours. The exosomes are purified by dialysis or immunoaffinity method; Step 2: Quantitative analysis of the extracted exosomes, the concentration of the exosomes is 10^9 to 10^12 particles / ml, preferably 10^10 particles / ml; Step 3: injecting the exosomes into the fracture site of the diabetic patient by intravenous injection, local injection or minimally invasive injection, with an injection dose of 10^10 particles / ml, once a week, and continuing the treatment for 4 to 6 weeks; Step 4: The exosome dose for each injection is 10^10 particles / ml, once a week, and the treatment lasts for 4-6 weeks. The specific treatment cycle is adjusted appropriately according to the patient's healing progress; Step 5: Evaluate the progress of fracture healing in diabetic patients through imaging examinations and bone density measurements, and adjust the treatment plan based on the examination results.

2. The method for promoting diabetic fracture healing by bone marrow mesenchymal stem cell exosomes according to claim 1, characterized in that: The exosome extraction method is to obtain exosomes by ultra-high-speed centrifugation, purify them by dialysis or immunoaffinity, and the purity reaches more than 85%, ensuring that the exosomes will not be interfered by cell debris and other contaminants during the extraction process.

3. The method for promoting diabetic fracture healing by bone marrow mesenchymal stem cell exosomes according to claim 1, characterized in that: The exosomes contain one of miRNA or mRNA, and the miRNA includes miR-21 and miR-29b. The miRNA or mRNA enters the target cells through the transport of exosomes. The miRNA or mRNA is used to intervene in the cell behavior at the fracture site. The miRNA and mRNA are selected based on their role in bone and soft tissue repair.

4. The method for promoting diabetic fracture healing by bone marrow mesenchymal stem cell exosomes according to claim 1, characterized in that: The concentration of the exosomes ranges from 10^9 to 10^12 particles / ml, and the concentration of the exosomes is adjusted according to the clinical manifestations and course of the diabetic patient.

5. The method for promoting diabetic fracture healing by bone marrow mesenchymal stem cell exosomes according to claim 1, characterized in that: The exosomes are injected into the fracture site of the diabetic patient by intravenous injection, local injection or minimally invasive injection. The local injection uses a fine needle to precisely inject the exosomes into the fracture area, and the minimally invasive injection assists the delivery of the exosomes by using a biocompatible carrier.

6. The method for promoting diabetic fracture healing by bone marrow mesenchymal stem cell exosomes according to claim 1, characterized in that: During the treatment process, dynamic imaging examinations combined with bone density tests and fracture healing assessment indicators are used to monitor the bone healing status in real time to guide the adjustment of treatment plans and ensure that the use of exosomes is compatible with the patient's fracture healing process.

7. The method for promoting diabetic fracture healing by bone marrow mesenchymal stem cell exosomes according to claim 1, characterized in that: The miRNA or mRNA in the exosomes can interact with target cells through the cell membrane during the treatment process, activating osteoblasts and vascular endothelial cells.

8. The method for promoting diabetic fracture healing by bone marrow mesenchymal stem cell exosomes according to claim 1, characterized in that: The carrier of the exosomes is selected to be liposomes, nanoparticles or other biocompatible materials.

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