Bone joint repairing composition containing stem cell vesicles and application thereof

Abstract

The invention discloses a bone joint repairing composition containing stem cell vesicles and application of the bone joint repairing composition, and relates to the technical field of bone joint repairing compositions.The bone joint repairing composition is prepared from, by weight, 90-110 parts of stem cell vesicles and 41-57 parts of thelenota ananas saponin Ananaside A. The stem cell vesicles and the thelenota ananas saponin Ananaside A are combined to form the bone joint repairing composition. The stem cell vesicles are extracellular vesicles derived from human amniotic mesenchymal stem cells. According to the bone joint repairing composition provided by the invention, the stem cell vesicles and the thelenota ananas saponin in a proper proportion are matched for use, so that the synergistic interaction between the stem cell vesicles and the thelenota ananas saponin is realized. Animal experiments show that the composition has a remarkable repairing effect on a rat model with bone and joint injuries, can effectively relieve bone and joint pain symptoms of rats, not only widens the application field of human amniotic mesenchymal stem cell-derived extracellular vesicles, but also provides a new clinical treatment means for bone and joint repairing.

Description

Technical Field

[0001] The present invention relates to the technical field of bone and joint repair compositions, and in particular to a bone and joint repair composition containing stem cell vesicles and applications thereof. Background Art

[0002] Bone and joint diseases, as a broad medical problem that includes osteoarthritis, joint injuries, degenerative changes and other complex diseases, have long had a serious impact on the daily lives of a large number of patients. With the continuous aging of the global population and the frequent occurrence of sports injuries, exploring efficient and safe methods for bone and joint repair has become a top priority in the field of medical research. Traditional treatments, such as drug therapy, physical therapy and surgical intervention, although they can relieve patients' symptoms to a certain extent, are often accompanied by many limitations such as obvious side effects, long recovery period and limited treatment effect.

[0003] In recent years, stem cell therapy has gradually emerged in the field of treatment of bone and joint diseases due to its potential for regeneration and repair, and has shown great application prospects. In particular, mesenchymal stem cells (MSCs) and their secreted extracellular vesicles (MSC-sEVs) have been confirmed by a large number of studies to have significant immunomodulatory functions. These extracellular vesicles can effectively induce macrophages to polarize to the M2 type, thereby promoting the repair and regeneration of defective bone tissue, providing a new idea for the treatment of inflammatory bone resorption diseases.

[0004] Among the many sources of MSCs, human amniotic mesenchymal stem cells (HAMSCs) have attracted much attention due to their unique advantages. HAMSCs are derived from the "discarded" amniotic tissue after childbirth. They are not only easy to obtain and widely available, but also do not involve ethical disputes. In addition, HAMSCs have significant characteristics such as low immunogenicity and strong multi-differentiation potential, which makes them have broad application potential in the fields of cell therapy and tissue engineering. However, although HAMSCs and their secreted extracellular vesicles (HAMSCs-sEV) have shown excellent performance in many aspects, there are currently no reports on the specific effects of HAMSCs-sEV in bone and joint repair.

[0005] In view of this, the present invention proposes a bone and joint repair composition containing stem cell vesicles and its application, aiming to explore its potential as a new bone and joint repair material through in-depth research on the effect of HAMSCs-sEV in bone and joint repair, and to provide a new, more efficient and safe clinical treatment plan for the treatment of bone and joint diseases. Summary of the invention

[0006] In order to solve the above problems, the present invention provides a bone and joint repair composition containing stem cell vesicles and its application.

[0007] In a first aspect, the present invention provides a bone and joint repair composition containing stem cell vesicles, wherein the bone and joint repair composition comprises the following components in parts by weight: 90-110 parts of stem cell vesicles and 41-57 parts of Ananaside A; The stem cell vesicles are extracellular vesicles derived from human amniotic mesenchymal stem cells; The chemical structural formula of the ginsenoside Ananaside A is shown in formula (I); The formula (I): .

[0008] Furthermore, the bone and joint repair composition comprises the following components in parts by weight: 100 parts of stem cell vesicles and 48.5 parts of Ananaside A.

[0009] Furthermore, the bone and joint repair composition comprises the following components in parts by weight: 110 parts of stem cell vesicles and 41 parts of Ananaside A, a ginsenoside from plum blossom ginseng.

[0010] Furthermore, the bone and joint repair composition comprises the following components in parts by weight: 90 parts of stem cell vesicles and 57 parts of Ananaside A.

[0011] Furthermore, the preparation method of the bone and joint repair composition comprises the following steps: Obtaining stem cell vesicles; The ginsenoside Ananaside A is loaded into the stem cell vesicles to obtain the bone and joint repair composition.

[0012] Furthermore, the loading method includes incubation, electroporation or extrusion.

[0013] Furthermore, the step of loading Ananaside A into the stem cell vesicles to obtain the bone and joint repair composition comprises the following process: The Ananaside A and the stem cell vesicles are mixed and incubated at 36-38°C for 2-4 hours, followed by a first centrifugation at 10000-15000g for 5-10 minutes and a second centrifugation at 500-1000g for 5-10 minutes to obtain the bone and joint repair composition.

[0014] In a second aspect, the present invention provides a use of a bone and joint repair composition containing stem cell vesicles as described in any one of the first aspects in the preparation of a drug for treating bone and joint repair.

[0015] Furthermore, the dosage form of the drug includes tablets, capsules, powders, mixtures, pills or granules.

[0016] The above technical solution provided by the embodiment of the present invention has at least the following advantages compared with the prior art: The embodiment of the present invention provides a bone and joint repair composition containing stem cell vesicles and its application. The bone and joint repair composition provided by the present invention is used in combination with stem cell vesicles and ginsenosides in an appropriate proportion to achieve a synergistic effect between the two. Animal experiments have shown that the composition has a significant repair effect on a rat model of bone and joint injury and can effectively relieve the bone and joint pain symptoms of rats. It not only broadens the application field of extracellular vesicles derived from human amniotic mesenchymal stem cells, but also provides a new clinical treatment method for bone and joint repair. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate embodiments consistent with the invention and, together with the description, serve to explain the principles of the invention.

[0018] 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 or the description of the prior art will be briefly introduced below. Obviously, for ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative labor.

[0019] Figure 1 A schematic flow chart of a method for preparing a bone and joint repair composition containing stem cell vesicles provided in an embodiment of the present invention. DETAILED DESCRIPTION

[0020] In order to make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are 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.

[0021] Unless otherwise specified, various raw materials, reagents, instruments and equipment used in the present invention can be purchased from the market or prepared by existing methods.

[0022] In a first aspect, the present invention provides a bone and joint repair composition containing stem cell vesicles, wherein the bone and joint repair composition comprises the following components in parts by weight: 90-110 parts of stem cell vesicles and 41-57 parts of Ananaside A; The stem cell vesicles are extracellular vesicles derived from human amniotic mesenchymal stem cells; The chemical structural formula of the ginsenoside Ananaside A is shown in formula (I); The formula (I): .

[0023] The embodiment of the present invention provides a bone and joint repair composition containing stem cell vesicles. The bone and joint repair composition provided by the present invention is used in combination with stem cell vesicles and ginsenosides in an appropriate proportion to achieve a synergistic effect between the two. Animal experiments have shown that the composition has a significant repair effect on a rat model of bone and joint injury and can effectively relieve the bone and joint pain symptoms of rats. It not only broadens the application field of extracellular vesicles derived from human amniotic mesenchymal stem cells, but also provides a new clinical treatment method for bone and joint repair.

[0024] The extracellular vesicles derived from human amniotic mesenchymal stem cells in the present invention can be directly commercially available products or can be prepared in-house according to the preparation methods disclosed in the prior art such as CN118207158A and CN111394305A, which will not be described in detail here.

[0025] The ginsenoside Ananaside A in the present invention is disclosed in prior art such as CN100537595C, and can be directly obtained from a commercial product or prepared in-house according to the preparation process disclosed in prior art such as CN100537595C, which will not be described in detail here.

[0026] In some specific embodiments, the bone joint repair composition comprises the following components in parts by weight: 100 parts of stem cell vesicles and 48.5 parts of Ananaside A.

[0027] In some specific embodiments, the bone joint repair composition comprises the following components in parts by weight: 110 parts of stem cell vesicles and 41 parts of Ananaside A, a ginsenoside from plum blossom ginseng.

[0028] In some specific embodiments, the bone joint repair composition comprises the following components in parts by weight: 90 parts of stem cell vesicles and 57 parts of Ananaside A.

[0029] In some specific embodiments, Figure 1 As shown, the preparation method of the bone joint repair composition comprises the following steps: Obtaining stem cell vesicles; The ginsenoside Ananaside A is loaded into the stem cell vesicles to obtain the bone and joint repair composition.

[0030] In some specific embodiments, the loading method includes incubation, electroporation or extrusion.

[0031] In some specific embodiments, the step of loading Ananaside A into the stem cell vesicles to obtain the bone and joint repair composition comprises the following process: The Ananaside A and the stem cell vesicles are mixed and incubated at 36-38°C for 2-4 hours, followed by a first centrifugation at 10000-15000g for 5-10 minutes and a second centrifugation at 500-1000g for 5-10 minutes to obtain the bone and joint repair composition.

[0032] In a second aspect, based on the same inventive concept, the present invention provides a use of a bone and joint repair composition containing stem cell vesicles as described in any one of the first aspects in the preparation of a drug for treating bone and joint repair.

[0033] In some specific embodiments, the dosage form of the drug includes tablets, capsules, powders, mixtures, pills or granules.

[0034] It should be noted that the bone and joint repair composition containing stem cell vesicles and the component raw materials involved in its application provided by the embodiments of the present invention, unless otherwise specified or specified, can be directly commercially available products or homemade using existing public preparation methods; at the same time, the steps and parameters involved, unless otherwise specified or specified, can be carried out according to the process steps and parameters disclosed in the prior art or directly carried out using existing equipment, and the present invention document will not describe them one by one.

[0035] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are intended only to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods in the following examples that do not specify specific conditions are usually measured according to national standards. If there is no corresponding national standard, then the conditions recommended by the manufacturer are followed.

[0036] The extracellular vesicles derived from human amniotic mesenchymal stem cells in the following Examples 1 to 3, Comparative Example 1 and Comparative Example 3 are prepared according to the preparation method disclosed in the prior art, specifically comprising the following steps: (1) culturing human amniotic mesenchymal stem cells of passage 3 to 5 with α-MEM complete medium containing 15% fetal bovine serum. When the density reaches 75%, the medium is discarded, the cells are washed 3 to 5 times with PBS, and the medium is replaced with α-MEM (without phenol red) culture medium containing 5% fetal bovine serum without extracellular vesicles. After continuing to culture for 48 hours, the cell culture supernatant is collected; (2) 10 ml of the cell culture supernatant is taken, first centrifuged at 300g and 4°C for 10 min, then centrifuged at 2000g and 4°C for 10 min, and finally centrifuged at 10000g and 4°C for 30 min, the supernatant is taken, the cell debris is removed using a 0.22 μm pore size filter, and the cell culture supernatant is centrifuged at 10000g and 4°C for 10 min to obtain the extracellular vesicles derived from human amniotic mesenchymal stem cells.

[0037] The preparation method of the bone and joint repair composition containing stem cell vesicles in the following Examples 1 to 3 and Comparative Example 3 comprises the following steps: Ananaside A and stem cell vesicles (specifically extracellular vesicles derived from human amniotic mesenchymal stem cells) are mixed and incubated at 37.5°C for 3.5 hours, followed by a first centrifugation at 12000g for 6 minutes and a second centrifugation at 700g for 8 minutes to obtain the bone and joint repair composition.

[0038] The chemical structural formula of Ananaside A in the following Examples 1 to 3, Comparative Example 2 and Comparative Example 3 is shown in Formula (I); The formula (I): .

[0039] Example 1 This example provides a bone and joint repair composition containing stem cell vesicles, which comprises the following components in parts by weight: 100 parts of stem cell vesicles and 48.5 parts of Ananaside A; The stem cell vesicles are extracellular vesicles derived from human amniotic mesenchymal stem cells.

[0040] Example 2 This example provides a bone and joint repair composition containing stem cell vesicles, which comprises the following components in parts by weight: 90 parts of stem cell vesicles and 57 parts of Ananaside A; The stem cell vesicles are extracellular vesicles derived from human amniotic mesenchymal stem cells.

[0041] Example 3 This example provides a bone and joint repair composition containing stem cell vesicles, which comprises the following components in parts by weight: 110 parts of stem cell vesicles and 41 parts of Ananaside A; The stem cell vesicles are extracellular vesicles derived from human amniotic mesenchymal stem cells.

[0042] Comparative Example 1 This example provides a bone and joint repair material, which is an extracellular vesicle derived from human amniotic mesenchymal stem cells.

[0043] Comparative Example 2 This example provides a bone and joint repair material, wherein the bone and joint repair material is Ananaside A, a ginsenoside of plum blossom.

[0044] Comparative Example 3 This example provides a bone and joint repair composition containing stem cell vesicles, which comprises the following components in parts by weight: 100 parts of stem cell vesicles and 100 parts of Ananaside A; The stem cell vesicles are extracellular vesicles derived from human amniotic mesenchymal stem cells.

[0045] Test Case In this example, the repair effects of the bone and joint repair compositions containing stem cell vesicles obtained in Examples 1 to 3 and Comparative Example 3 and the bone and joint repair materials obtained in Comparative Examples 1 and 2 on a rat model of bone and joint injury were investigated according to the test methods disclosed in the prior art.

[0046] Test method: 1) Modeling: SD rats were anesthetized with 2% chloral hydrate to reduce stress during surgery; then the hair at the knee joint of the right leg of the rat was gently removed with a depilatory agent to ensure that the surgical area was sterile to prevent infection and facilitate surgical operations. Under sterile conditions, the skin was incised and the subcutaneous tissue was separated layer by layer, and the surrounding blood vessels and nerves were carefully separated and protected. After exposing the articular surface and the medial meniscus, the medial meniscus was accurately removed, and the anterior and posterior cruciate ligaments were carefully cut to simulate bone and joint injury. During the operation, attention should be paid to hemostasis to avoid postoperative infection; when suturing the subcutaneous connective tissue and skin, ensure that the wound is well aligned to reduce scar formation; after the operation, the rat is placed in a suitable environment, and it is fully awake and restored to activity to obtain a rat bone and joint injury model; 2) Administration test: The rat bone and joint injury model is administered with the bone and joint repair composition containing stem cell vesicles obtained in Examples 1 to 3 and Comparative Example 3, and the bone and joint repair material obtained in Comparative Example 1 and Comparative Example 2, respectively, 10 rats in each group, and the administration is given once a day by oral gavage in the morning and evening, each administration is 5 mg / kg, and the administration is continued for 14 days. 3) The pain score of rats before and after treatment is evaluated by spontaneous pain behavior: Preparation before evaluation: Ensure that the evaluation environment is quiet, undisturbed, and the temperature is appropriate to avoid the influence of external factors on the behavior of rats; Evaluation before treatment: Observe the behavior of rats during free activities, especially the activity of the right knee joint. According to the degree of pain shown by the rats during activities, the scoring standard of 0 to 10 points (as shown in Table 1) is used for scoring (the more obvious the pain symptoms, the higher the score value (S)). Record the activity pain value of each rat before treatment. Post-treatment evaluation: 14 days after administration, observe the behavior of the rats during free activities again. Use the same scoring criteria to score the pain level of the rats during activities after treatment. Record the activity pain value after treatment for each rat. Calculate the activity pain reduction value: According to the formula: Activity pain reduction value = Activity pain value before treatment - Activity pain value after treatment, calculate the activity pain reduction value of each rat. The greater the activity pain reduction value, the better the repair effect on the rat model of bone and joint injury. The test results are shown in Table 2.

[0047] Table 1 Table 2 It can be seen from Table 2 that compared with comparative examples 1 to 3, the bone and joint repair compositions containing stem cell vesicles provided in Examples 1 to 3 of the present invention have better repair effects on the bone and joint injury rat model, and can effectively alleviate the bone and joint pain symptoms of rats; and the bone and joint repair compositions provided by the present invention are used in combination with stem cell vesicles and ginsenosides in appropriate proportions, and the two show obvious synergistic effects.

[0048] In summary, the bone and joint repair composition provided by the present invention is used in combination with stem cell vesicles and ginsenosides in an appropriate ratio to achieve a synergistic effect between the two. Animal experiments have shown that the composition has a significant repair effect on the bone and joint injury rat model and can effectively relieve the bone and joint pain symptoms of rats. It not only broadens the application field of extracellular vesicles derived from human amniotic mesenchymal stem cells, but also provides a new clinical treatment method for bone and joint repair.

[0049] Various embodiments of the present invention may be presented in the form of a range; it should be understood that the description in the form of a range is only for convenience and brevity, and should not be understood as a rigid limitation on the scope of the present invention; therefore, the range description should be considered to have specifically disclosed all possible sub-ranges and single numerical values ​​within the range. For example, the range description from 1 to 6 should be considered to have specifically disclosed sub-ranges, such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., as well as single numbers within the range, such as 1, 2, 3, 4, 5 and 6, which apply regardless of the range. In addition, whenever a numerical range is indicated herein, it is meant to include any cited number (fractional or integer) within the indicated range.

[0050] The foregoing is merely a specific embodiment of the present invention, which enables those skilled in the art to understand or implement the present invention. Various modifications to these embodiments will be apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but rather to the widest scope consistent with the principles and novel features claimed herein.

Claims

1. A bone and joint repair composition containing stem cell vesicles, characterized in that: The bone and joint repair composition comprises the following components in parts by weight: 90-110 parts of stem cell vesicles and 41-57 parts of Ananaside A; The stem cell vesicles are extracellular vesicles derived from human amniotic mesenchymal stem cells; The chemical structural formula of the ginsenoside Ananaside A is shown in formula (I); The formula (I): .

2. The bone and joint repair composition containing stem cell vesicles according to claim 1, characterized in that: The bone and joint repair composition comprises the following components in parts by weight: 100 parts of stem cell vesicles and 48.5 parts of Ananaside A.

3. The bone and joint repair composition containing stem cell vesicles according to claim 1, characterized in that: The bone and joint repair composition comprises the following components in parts by weight: 110 parts of stem cell vesicles and 41 parts of Ananaside A, a ginsenoside from plum blossom ginseng.

4. The bone and joint repair composition containing stem cell vesicles according to claim 1, characterized in that: The bone and joint repair composition comprises the following components in parts by weight: 90 parts of stem cell vesicles and 57 parts of Ananaside A.

5. The bone and joint repair composition containing stem cell vesicles according to any one of claims 1 to 4, characterized in that: The preparation method of the bone and joint repair composition comprises the following steps: Obtaining stem cell vesicles; The ginsenoside Ananaside A is loaded into the stem cell vesicles to obtain the bone and joint repair composition.

6. The bone and joint repair composition according to claim 5, characterized in that: The loading method includes incubation, electroporation or extrusion.

7. The bone and joint repair composition according to claim 5, characterized in that: The step of loading Ananaside A into the stem cell vesicles to obtain the bone and joint repair composition comprises the following process: The Ananaside A and the stem cell vesicles are mixed and incubated at 36-38°C for 2-4 hours, followed by a first centrifugation at 10000-15000g for 5-10 minutes and a second centrifugation at 500-1000g for 5-10 minutes to obtain the bone and joint repair composition.

8. Use of the bone and joint repair composition containing stem cell vesicles according to any one of claims 1 to 7 in the preparation of a drug for treating bone and joint repair.

9. The use according to claim 8, characterized in that: The dosage form of the drug includes tablets, capsules, powders, mixtures, pills or granules.

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