Use of a pharmaceutical composition in promoting osteogenic differentiation of mesenchymal stem cells

Abstract

The application provides application of a pharmaceutical composition in promoting mesenchymal stem cell osteogenic differentiation, and the pharmaceutical composition comprises metformin and forskolin. The application creatively combines metformin and forskolin, and compared with metformin or forskolin alone, can more significantly promote mesenchymal stem cell osteogenic differentiation, does not need to add traditional osteogenic inducer, can significantly improve the expression amount of ALP, OCN and RUNX2 at 14 days, and obvious calcium deposition phenomenon appears, thereby providing a basis for developing an effective new drug combination strategy in the field of bone tissue engineering.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to the application of a pharmaceutical composition in promoting osteogenic differentiation of mesenchymal stem cells. Background Technology

[0002] Mesenchymal stem cells (MSCs) are a valuable biological resource capable of self-renewal, immune regulation, and angiogenesis promotion. MSCs possess the potential to differentiate into multiple lineages, including osteoblasts, chondrocytes, adipocytes, and neurons, and exhibit potent immunomodulatory and anti-inflammatory effects. Simultaneously, by secreting cytokines and exosomes, they exert immunomodulatory and paracrine effects, inhibiting inflammatory responses and promoting angiogenesis and tissue repair. Their low immunogenicity and high proliferative stability make them suitable for allogeneic transplantation, demonstrating significant potential in the treatment of cardiovascular diseases, nerve injuries, bone and joint repair, and diabetes.

[0003] Osteoblasts play a central role in bone formation and repair, leading bone tissue construction and regeneration after injury by synthesizing bone matrix (such as collagen) and promoting its mineralization. However, current methods for promoting osteoblast differentiation, such as biomaterial scaffolds, growth factors, gene editing technologies, and small molecule drugs, still face significant limitations. Growth factors are expensive and prone to causing side effects such as ectopic ossification; gene therapy may pose a carcinogenic risk due to the use of viral vectors; insufficient biocompatibility and mechanical properties of biomaterials may affect osteogenic differentiation efficiency; and long-term drug use carries metabolic burdens and off-target effects.

[0004] In addition, existing technologies generally suffer from problems such as low differentiation efficiency, difficulty in standardized preparation, and long clinical translation cycles, and there is an urgent need to develop safer, more economical, and more precise regulatory strategies to optimize the efficacy of bone regeneration. Summary of the Invention

[0005] To address the shortcomings of existing technologies, the present invention aims to provide an application of a pharmaceutical composition in promoting osteogenic differentiation of mesenchymal stem cells. The combined use of metformin and forscolin can significantly promote osteogenic differentiation of mesenchymal stem cells, providing a basis for developing effective new drug combination strategies in the field of bone tissue engineering.

[0006] To achieve this objective, the present invention adopts the following technical solution:

[0007] In a first aspect, the present invention provides the use of a pharmaceutical composition for promoting osteogenic differentiation of mesenchymal stem cells for purposes other than disease diagnosis and / or treatment, said pharmaceutical composition comprising metformin and forscoringin.

[0008] Metformin is a primary treatment for type 2 diabetes and also plays an important role in various biological processes, including anti-tumor, anti-inflammatory, anti-aging, cardiac and hepatic protection, and tissue regeneration. Metformin not only promotes the differentiation of induced pluripotent stem cells (iPSCs) into osteoblasts, but also enhances their mineralization capacity, protects osteoblasts from damage caused by high glucose environments, and directly promotes osteoblast proliferation. It also regulates stem cell metabolism and differentiation by activating the AMPK / mTOR signaling pathway.

[0009] Fuscolin is a natural diterpenoid compound extracted from Coleus formosanus. It can directly activate adenylate cyclase, thereby increasing the level of intracellular cyclic adenosine monophosphate (cAMP). As an important second messenger for intracellular signal transduction, cAMP can affect a variety of cellular activities, such as regulating lipolysis, glycogenolysis, myocardial contraction, smooth muscle relaxation, and inhibiting platelet aggregation. It also has a certain regulatory effect on processes such as cell differentiation.

[0010] This invention creatively discovers that the combined use of metformin and forscolin can significantly promote the osteogenic differentiation of mesenchymal stem cells, providing a foundation for developing effective novel drug combination strategies in the field of bone tissue engineering. In this invention, the combined use of metformin and forscolin, compared to metformin or forscolin alone, can more significantly promote the osteogenic differentiation of mesenchymal stem cells, exhibiting a synergistic effect.

[0011] This invention combines metformin and forscoringin to promote osteogenic differentiation of mesenchymal stem cells and applies it to basic research in the field of bone tissue engineering, such as constructing osteogenic differentiation models of mesenchymal stem cells and studying cell pathways related to osteogenic differentiation of mesenchymal stem cells.

[0012] Preferably, the molar ratio of metformin to forscoringin is (5-500):1.

[0013] The specific point values ​​in (5-500) can be 5, 8, 10, 15, 20, 50, 80, 100, 150, 200, 300, 400 or 500, etc.

[0014] Other specific point values ​​within the range of the above values ​​can be selected, and will not be elaborated on here.

[0015] In this invention, metformin and forscoringin are used in combination in the above-mentioned specific ratio. After 14 days, they can significantly promote the expression of osteogenic-related genes alkaline phosphatase (ALP), osteocalcin (OCN) and RUNX2, resulting in obvious calcium deposition.

[0016] Preferably, the molar ratio of metformin to forscoringin is (10-300):1.

[0017] The specific point values ​​in (10-300) can be 10, 12, 15, 18, 20, 30, 50, 80, 100, 200 or 300, etc.

[0018] Other specific point values ​​within the range of the above values ​​can be selected, and will not be elaborated on here.

[0019] In this invention, metformin and forscoringin are used in combination in the above-mentioned specific ratio. After 14 days, the expression levels of osteogenic-related genes ALP, OCN and RUNX2 are higher, and the calcium deposition phenomenon is more significant.

[0020] Preferably, the dosage of metformin is 0.1-10 mM, for example, 0.1 mM, 0.2 mM, 0.5 mM, 0.8 mM, 1 mM, 2 mM, 5 mM, 8 mM or 10 mM.

[0021] Preferably, the application concentration of forscorin is 5-20 μM, for example, 5 μM, 8 μM, 10 μM, 12 μM, 15 μM, 18 μM or 20 μM.

[0022] Other specific point values ​​within the range of the above values ​​can be selected, and will not be elaborated on here.

[0023] In this invention, metformin and forscoringin were used in combination at the above-mentioned specific concentrations. After 14 days, the expression levels of osteogenic-related genes ALP, OCN and RUNX2 were higher, and the calcium deposition phenomenon was more significant.

[0024] Preferably, the mesenchymal stem cells include any one or a combination of at least two of umbilical cord mesenchymal stem cells, bone marrow mesenchymal stem cells, adipose mesenchymal stem cells, placental mesenchymal stem cells, or synovial mesenchymal stem cells.

[0025] Preferably, the pharmaceutical composition is a single compound preparation or a combination of two separate preparations.

[0026] Preferably, the pharmaceutical composition is a combination of two separate formulations, which are administered simultaneously or sequentially.

[0027] It is understood that metformin and forscorin in the pharmaceutical composition can exist independently and can be administered simultaneously or sequentially to the target subject.

[0028] Preferably, the formulation is any pharmaceutically acceptable dosage form.

[0029] In a second aspect, the present invention provides the use of a pharmaceutical composition in the preparation of a medicament for the prevention or treatment of osteoporosis, said pharmaceutical composition comprising metformin and forscoringin.

[0030] Preferably, the molar ratio of metformin to forscoringin is (5-500):1.

[0031] The specific point values ​​in (5-500) can be 5, 8, 10, 15, 20, 50, 80, 100, 150, 200, 300, 400 or 500, etc.

[0032] Other specific point values ​​within the range of the above values ​​can be selected, and will not be elaborated on here.

[0033] Preferably, the molar ratio of metformin to forscoringin is (10-300):1.

[0034] The specific point values ​​in (10-300) can be 10, 12, 15, 18, 20, 30, 50, 80, 100, 200 or 300, etc.

[0035] Other specific point values ​​within the range of the above values ​​can be selected, and will not be elaborated on here.

[0036] Thirdly, the present invention provides the use of a pharmaceutical composition in the preparation of a medicament for treating bone defects, said pharmaceutical composition comprising metformin and forscolin.

[0037] Preferably, the molar ratio of metformin to forscoringin is (5-500):1.

[0038] The specific point values ​​in (5-500) can be 5, 8, 10, 15, 20, 50, 80, 100, 150, 200, 300, 400 or 500, etc.

[0039] Other specific point values ​​within the range of the above values ​​can be selected, and will not be elaborated on here.

[0040] Preferably, the molar ratio of metformin to forscoringin is (10-300):1.

[0041] The specific point values ​​in (10-300) can be 10, 12, 15, 18, 20, 30, 50, 80, 100, 200 or 300, etc.

[0042] Other specific point values ​​within the range of the above values ​​can be selected, and will not be elaborated on here.

[0043] Fourthly, the present invention provides the use of a pharmaceutical composition in the preparation of a mesenchymal stem cell osteogenic inducer, said pharmaceutical composition comprising metformin and forscolin.

[0044] Preferably, the molar ratio of metformin to forscoringin is (5-500):1.

[0045] The specific point values ​​in (5-500) can be 5, 8, 10, 15, 20, 50, 80, 100, 150, 200, 300, 400 or 500, etc.

[0046] Other specific point values ​​within the range of the above values ​​can be selected, and will not be elaborated on here.

[0047] Preferably, the molar ratio of metformin to forscoringin is (10-300):1.

[0048] The specific point values ​​in (10-300) can be 10, 12, 15, 18, 20, 30, 50, 80, 100, 200 or 300, etc.

[0049] Other specific point values ​​within the range of the above values ​​can be selected, and will not be elaborated on here.

[0050] Preferably, the mesenchymal stem cells include any one or a combination of at least two of umbilical cord mesenchymal stem cells, bone marrow mesenchymal stem cells, adipose mesenchymal stem cells, placental mesenchymal stem cells, or synovial mesenchymal stem cells.

[0051] Compared with the prior art, the present invention has the following beneficial effects:

[0052] This invention creatively combines metformin and forscoringin, which significantly promotes osteogenic differentiation of mesenchymal stem cells compared to metformin or forscoringin alone. It does not require the addition of traditional osteogenic inducers and can significantly increase the expression levels of ALP, OCN, and RUNX2 within 14 days, resulting in obvious calcium deposition. This provides a basis for developing effective new drug combination strategies in the field of bone tissue engineering. Attached Figure Description

[0053] Figure 1 This is a comparison of the Western blot results of human umbilical cord mesenchymal stem cells 14 days after osteogenic induction in Example 1 and Comparative Example 3.

[0054] Figure 2 Alizarin Red staining image of human umbilical cord mesenchymal stem cells 14 days after osteogenic induction in Example 1;

[0055] Figure 3 Alizarin Red staining image of human umbilical cord mesenchymal stem cells 14 days after osteogenic induction in Example 2;

[0056] Figure 4 Alizarin Red staining image of human umbilical cord mesenchymal stem cells 14 days after osteogenic induction in Example 3. Detailed Implementation

[0057] To further illustrate the technical means and effects of the present invention, the following describes the technical solution of the present invention in conjunction with preferred embodiments of the present invention. However, the present invention is not limited to the scope of the embodiments.

[0058] Where specific techniques or conditions are not specified in the examples, they shall be performed in accordance with the techniques or conditions described in the literature in this field, or in accordance with the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased through legitimate channels.

[0059] The metformin mentioned in the following examples is from MCE (catalog number: HY-B0627), and the foscorlin is from Xili Bio (catalog number: 66575-29-9).

[0060] Extraction and culture of human umbilical cord mesenchymal stem cells: The umbilical cord (derived from the Zhejiang Provincial Umbilical Cord Blood Bank) was sterilized in 70% ethanol for 40 seconds, then rinsed five times with 0.9% saline, ensuring complete immersion of the umbilical cord in saline during each rinse. After rinsing, the umbilical vein (the largest opening in the cross-section of the umbilical cord) was cut open with surgical scissors. A layer of epithelial cells on the inner wall of the cut vein was gently peeled off with tissue forceps. The two arteries were then separated and torn open along their periphery with tissue forceps. The separated umbilical cord tissue was placed in a 50mL centrifuge tube containing 10mL of complete culture medium. The umbilical cord tissue in the centrifuge tube was cut into 1mm pieces of a paste using surgical scissors. The tissue blocks were then aspirated using a Pasteur pipette and placed into a T25 culture flask. An appropriate amount of complete culture medium was added to the culture flask. The umbilical cord tissue blocks were first gathered in the lower right corner of the culture flask, then spread evenly along the right side. The flask was then shaken rapidly to ensure the tissue blocks were evenly distributed at the bottom. The flask was cultured at 37℃ and 5% CO2. When the cell confluence reached 90%, the cells were digested with trypsin and set aside for later use.

[0061] The complete culture medium contains 94% MSCBM (Type B) basal medium (from Shenzhen Dako Biotechnology Co., Ltd.) and 5% serum substitute EliteGro. TM -Adv (GMP) (from Biomedical EliteCell Corp.) and 1% penicillin / streptomycin (from HCMbiotech).

[0062] Example 1

[0063] 1×10⁶ cells were seeded in a 12-well plate. 6Human umbilical cord mesenchymal stem cells (MCSCs) were cultured at 37°C and 5% CO2 for 14 days. Metformin and forscorin were added to the complete culture medium to induce osteogenic activity in the MCSCs: metformin was administered at a concentration of 0.1 mM at the start of culture, once a day; forscorin was administered at a concentration of 10 μM on day 3 of culture, once every two days.

[0064] Example 2

[0065] 1×10⁶ cells were seeded in a 12-well plate. 6 Human umbilical cord mesenchymal stem cells (MCSCs) were cultured at 37°C and 5% CO2 for 14 days. Metformin and forscorin were added to the complete culture medium to induce osteogenic activity in the MCSCs: metformin was administered at a concentration of 1 mM at the start of culture, once a day; forscorin was administered at a concentration of 20 μM on day 3 of culture, once every two days.

[0066] Example 3

[0067] 1×10⁶ cells were seeded in a 12-well plate. 6 Human umbilical cord mesenchymal stem cells (MCSCs) were cultured at 37°C and 5% CO2 for 14 days. Metformin and forscorin were added to the complete culture medium to induce osteogenic activity in the MCSCs: metformin was added at a concentration of 1.5 mM at the start of culture once a day; forscorin was added at a concentration of 5 μM on day 3 of culture once every two days.

[0068] Example 4

[0069] 1×10⁶ cells were seeded in a 12-well plate. 6 Human umbilical cord mesenchymal stem cells (MCSCs) were cultured at 37°C and 5% CO2 for 14 days. Metformin and forscoringin were added to the complete culture medium to induce osteogenic activity in the MCSCs: metformin was administered at a concentration of 5 mM at the start of culture, once a day; forscoringin was administered at a concentration of 10 μM on day 3 of culture, once every two days.

[0070] Example 5

[0071] 1×10⁶ cells were seeded in a 12-well plate. 6Human umbilical cord mesenchymal stem cells (MCSCs) were cultured at 37°C and 5% CO2 for 14 days. Metformin and forscorin were added to the complete culture medium to induce osteogenic activity in the MCSCs: metformin was administered at a concentration of 0.1 mM at the start of culture, once a day; forscorin was administered at a concentration of 20 μM on day 3 of culture, once every two days.

[0072] Example 6

[0073] 1×10⁶ cells were seeded in a 12-well plate. 6 Human umbilical cord mesenchymal stem cells (MCSCs) were cultured at 37°C and 5% CO2 for 14 days. Metformin and forscoringin were added to the complete culture medium to induce osteogenic activity in the MCSCs: metformin was administered at a concentration of 7 mM at the start of culture, once a day; forscoringin was administered at a concentration of 10 μM on day 3 of culture, once every two days.

[0074] Example 7

[0075] 1×10⁶ cells were seeded in a 12-well plate. 6 Human umbilical cord mesenchymal stem cells (MCSCs) were cultured at 37°C and 5% CO2 for 14 days. Metformin and forscorin were added to the complete culture medium to induce osteogenic activity in the MCSCs: metformin was administered at a concentration of 0.02 mM at the start of culture, once a day; forscorin was administered at a concentration of 20 μM on day 3 of culture, once every two days.

[0076] Comparative Example 1

[0077] 1×10⁶ cells were seeded in a 12-well plate. 6 Human umbilical cord mesenchymal stem cells were cultured at 37°C and 5% CO2 for 14 days. Metformin was added to the complete culture medium to induce osteogenic activity of the umbilical cord mesenchymal stem cells. The metformin concentration was 0.11 mM, added at the beginning of the culture, and the frequency of administration was once a day.

[0078] Comparative Example 2

[0079] 1×10⁶ cells were seeded in a 12-well plate. 6 Human umbilical cord mesenchymal stem cells were cultured at 37°C and 5% CO2 for 14 days. Osteogenic induction of umbilical cord mesenchymal stem cells was performed in complete culture medium with forscoring: the concentration of forscoring was 110 μM, added on day 3 of culture, and administered once every 2 days.

[0080] Comparative Example 3

[0081] 1×10⁶ cells were seeded in a 12-well plate. 6 Human umbilical cord mesenchymal stem cells (HMCs) were cultured at 37°C and 5% CO2 for 14 days. Osteogenic induction of HMCs was then induced by applying osteogenic medium every two days. The osteogenic medium formulation consisted of 50 μM ascorbic acid, 100 nM dexamethasone, and 10 mM sodium β-glycerophosphate.

[0082] Test Example 1 Osteogenesis Markers

[0083] After culturing human umbilical cord mesenchymal stem cells for 14 days in Examples 1-7 and Comparative Examples 1-3, the cells were gently washed three times with pre-cooled physiological saline to remove culture medium and impurities. RIPA lysis buffer (containing protease inhibitors and phosphatase inhibitors) was added, and the cells were gently scraped off with a cell scraper and transferred to centrifuge tubes. The cells were incubated on ice for about 30 minutes to allow for complete cell lysis, with the centrifuge tubes gently tapped during this period to promote lysis. The lysed cell suspension was centrifuged at 12,000 rpm for 10 minutes at 4°C, and the supernatant was used to determine the protein concentration using a BCA protein quantification kit (from Nanjing Novizan Biotechnology Co., Ltd.).

[0084] Western blot assay: Samples with determined protein content were subjected to SDS-PAGE electrophoresis, followed by transfer to a PVDF membrane. After transfer, the PVDF membrane was removed from the transfer apparatus and placed in blocking buffer for blocking at room temperature for 1 hour. After blocking, the membrane was removed and gently washed three times with TBST buffer for 5 minutes each time to remove unbound blocking material. The membrane was then placed in primary antibody solution (antibody sources: ALP (Huaan Biotechnology, ET1601-21), OCN (Huaan Biotechnology, ER1919-20), RUNX2 (Huaan Biotechnology, ET1612-47)) and placed in a humidified chamber at 4°C. Incubate overnight to allow the primary antibody to specifically bind to the target protein. The next day, remove the membrane and wash it three times with TBST buffer for 10 minutes each time to thoroughly remove unbound primary antibody and reduce background signal. Place the membrane in the corresponding secondary antibody solution (antibody sources: ALP (Huaan Biotechnology, HA1001), OCN (Huaan Biotechnology, HA1001), RUNX2 (Huaan Biotechnology, HA1001)), gently shake on a shaker, and incubate at room temperature for 1 hour to allow the secondary antibody to specifically bind to the primary antibody. Remove the membrane and wash it three times with TBST buffer for 10 minutes each time. Finally, use an exposure instrument for color development, image acquisition, and quantitative analysis. Figure 1 This is a comparison of the Western blot results for osteogenic induction in Example 1 and Comparative Example 3.

[0085] The test results are shown in Table 1. This invention creatively combines metformin and forscolin, which significantly promotes osteogenic differentiation of mesenchymal stem cells compared to metformin or forscolin alone, without the need for traditional osteogenic inducing agents. It also significantly increases the expression levels of ALP, OCN, and RUNX2 within 14 days. Furthermore, the combined use of metformin and forscolin in a specific ratio demonstrates a more significant osteogenic induction effect on mesenchymal stem cells.

[0086] Table 1

[0087]

[0088]

[0089] Test Example 2: Calcium Deposition

[0090] Alizarin red staining was used to quantitatively analyze the calcium nodule content. Differentiation efficiency was defined as a ≥3-fold upregulation of marker expression and a mineralization area ratio >30%. After 14 days of culture of human umbilical cord mesenchymal stem cells in Examples 1-7 and Comparative Examples 1-3, the culture medium was removed, and the cells were gently rinsed three times with pre-cooled physiological saline. Then, 75% ethanol was added as a fixative to completely immerse the cells, and fixation was performed at room temperature for 15 minutes. The fixed cells exhibited relatively stable morphology and intracellular calcium salt precipitation, facilitating subsequent staining. The fixed cells were washed three times with physiological saline, and alizarin red staining solution was added, ensuring complete coverage of the cell layer. Staining was performed at room temperature in the dark for 30 minutes. After staining, the cells were gently rinsed with water to remove unbound alizarin red dye, rinsing three times until the background color was light and the red staining of the calcium nodules was clearly visible. The most representative example is the osteogenic-induced alizarin red staining effect in Examples 1-3, as shown below. Figure 2-4 As shown. Then, hexadecylpyridine chloride was used to dissolve the sample, and the solution was collected and the OD value was measured at 540 nm using a spectrophotometer (3 parallel samples were set up for each group, and each parallel sample was measured 3 times, and the final average value was taken).

[0091] The test results are shown in Table 2. Under a microscope, the morphology of cells undergoing osteogenic differentiation changed from elongated spindle-shaped to cuboidal or polygonal, and the pseudopodia shortened. This invention creatively combines metformin and forscolin, which, compared to metformin or forscolin alone, significantly promotes osteogenic differentiation of mesenchymal stem cells without the need for traditional osteogenic inducing agents, and noticeable calcium deposition appears after 14 days. Furthermore, the combined use of metformin and forscolin in a specific ratio has a more significant effect on inducing osteogenic differentiation of mesenchymal stem cells.

[0092] Table 2

[0093] Example 1 1.64 Example 2 1.80 Example 3 1.97 Example 4 1.38 Example 5 1.19 Example 6 1.18 Example 7 0.77 Comparative Example 1 0.63 Comparative Example 2 0.36 Comparative Example 3 0.90

[0094] This invention illustrates the application of a pharmaceutical composition of the present invention in promoting osteogenic differentiation of mesenchymal stem cells through the above embodiments. However, the present invention is not limited to the above embodiments, that is, it does not mean that the present invention must rely on the above embodiments to be implemented. Those skilled in the art should understand that any improvements to the present invention, equivalent substitutions of the raw materials of the product of the present invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.

[0095] The preferred embodiments of the present invention have been described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solution of the present invention, and these simple modifications all fall within the protection scope of the present invention.

[0096] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present invention will not describe the various possible combinations separately.

Claims

1. The use of a pharmaceutical composition in promoting osteogenic differentiation of mesenchymal stem cells for purposes other than disease diagnosis and treatment, characterized in that, The pharmaceutical composition comprises metformin and forscoringin, wherein the molar ratio of metformin to forscoringin is 10:1; the mesenchymal stem cells are umbilical cord mesenchymal stem cells.

2. The application according to claim 1, characterized in that, The pharmaceutical composition is a single compound preparation or a combination of two separate preparations.

3. The application according to claim 2, characterized in that, The pharmaceutical composition is a combination of two separate formulations, which are administered simultaneously or sequentially.

4. The application according to claim 2, characterized in that, The formulation can be any pharmaceutically acceptable dosage form.

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