Osteogenic differentiation induction medium based on pilose antler polypeptide and osteogenic differentiation induction method

Abstract

The invention discloses an osteogenic differentiation induction medium based on pilose antler polypeptide and an osteogenic differentiation induction method, and belongs to the technical field of biology. The osteogenic differentiation induction culture medium based on the cornu cervi pantotrichum polypeptide contains a cornu cervi pantotrichum polypeptide solution, dexamethasone, L-ascorbic acid, beta-sodium glycerophosphate and a complete culture medium, the cornu cervi pantotrichum polypeptide provided by the invention can greatly improve the osteogenic differentiation effect of mesenchymal stem cells as an additive component of an osteogenic induction culture medium, and is superior to a commercial osteogenic induction culture medium in a certain scene; according to the osteogenic induction differentiation method disclosed by the invention, the defects in the prior art are improved, and the osteogenic differentiation effect of the mesenchymal stem cells can be improved by adopting the pilose antler polypeptide osteogenic differentiation induction culture medium.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to an osteogenic differentiation induction culture medium and method based on deer antler polypeptide. Background Technology

[0002] Osteogenic induction medium is a specific culture system used to induce osteogenic stem cells, such as bone marrow mesenchymal stem cells (BMSCs), to differentiate into osteoblasts in vitro. It is an indispensable core tool in bone tissue engineering, bone regenerative medicine, and research on bone development mechanisms. Safe, stable, and effective osteogenic induction medium can improve the quality of in vitro osteogenic induction, provide reliable seed cells for bone tissue engineering, and is of great significance for bone defect repair and preclinical research.

[0003] Existing osteogenic induction media typically consist of a basal medium (such as α-MEM, DMEM, and F12 medium), fetal bovine serum (FBS), dexamethasone, L-ascorbic acid, and sodium β-glycerophosphate. Each component plays a different role in osteogenic induction: the basal medium provides the nutritional environment necessary for cell growth; FBS provides various growth factors and proteins to support early proliferation and adhesion; dexamethasone, as a glucocorticoid, promotes osteogenic differentiation through glucocorticoid receptor-mediated transcriptional regulation; L-ascorbic acid promotes type I collagen synthesis and extracellular matrix formation; and sodium β-glycerophosphate serves as a source of inorganic phosphorus required for organic matrix mineralization. Overall, existing osteogenic induction systems still face multiple bottlenecks in practical applications. First, the osteogenic induction effect exhibits significant instability, accompanied by lengthy induction cycles and low differentiation efficiency, resulting in high time costs for experimental and preclinical studies, making it difficult to meet the urgent need for efficient osteogenic induction. Secondly, existing osteogenic induction media have relatively simple compositions, which significantly limits their cross-species applicability. They often require the addition of specific growth factors or fine-tuning of formulation ratios based on the biological differences of cell types (e.g., human, mouse, or rabbit). This species-specific limitation results in a lack of universality in the media, making it difficult to develop a robust induction system that can stably function across species. Therefore, developing a stable, naturally synergistic, and universally applicable osteogenic induction media has become a critical issue that urgently needs to be addressed in the field of bone repair and regenerative medicine.

[0004] Deer antlers are the only organ in mammals capable of rapid, periodic regeneration and eventual ossification, growing at a rate of approximately 2 cm / day during their growth period. They ultimately form antlers, demonstrating a highly active ability to regulate osteogenic differentiation. Deer antler peptides, extracted from rapidly growing antlers, are a collection of factors that promote cell division and osteogenic differentiation. Through positive selection of osteogenic genes, they achieve a naturally optimal ratio of cytokines that synergistically promote bone regeneration. We first confirmed the osteogenic induction activity of deer antler peptides through in vitro cell experiments and optimized their concentration. Using this optimal concentration, we prepared an osteogenic induction medium for deer antler peptides. Then, by comparing the osteogenic induction activity with two other brands of mediums, we verified its advantages in improving osteogenic differentiation efficiency and cross-species application. Overall, the deer antler peptide osteogenic induction medium effectively shortens the osteogenic induction cycle, accelerates research progress, and has a wide range of applications, enabling cross-species (human and mouse) use. Summary of the Invention

[0005] The purpose of this invention is to solve the problems mentioned in the background art above, and to propose an osteogenic differentiation induction culture medium and an osteogenic differentiation induction method based on deer antler polypeptide.

[0006] An osteogenic differentiation induction culture medium based on deer antler polypeptide contains deer antler polypeptide solution, dexamethasone, L-ascorbic acid, sodium β-glycerophosphate and complete culture medium; The deer antler polypeptide solution is prepared by the following steps: S1. Cut fresh two-pronged deer antlers and peel off the skin. Cut the antler tissue into pieces to obtain small pieces of antler. S2. Place the small pieces of deer antler obtained in S1 into a centrifuge empty column tube, centrifuge to remove blood, and obtain small pieces of debloodened deer antler tissue. S3. The small pieces of deer antler tissue after blood removal in S2 are crushed to obtain deer antler tissue powder. S4. Mix the deer antler tissue powder obtained in S3 with water at a ratio of 1g:10-20mL, and add protease inhibitor to obtain a mixture; extract the mixture, filter it, and store the resulting extract separately. S5. Mix the solid deer antler tissue obtained from S4 with water at a ratio of 1g:10-20mL, add protease inhibitor, and obtain a mixture again. Extract the mixture, filter it, and store the resulting extract separately. Repeat this step several times. Mix all the above extracts to obtain a crude extract. S6. Filter the crude extract obtained in S5 using a filter membrane to obtain the filtrate; S7. Dialyze the filtrate obtained in S6 to remove inorganic salts and obtain a purified solution; S8. The purified solution obtained in S7 is freeze-dried to obtain a dried powder, which is the deer antler polypeptide extract. S9. The deer antler polypeptide extract obtained in S8 is resuspended and dissolved in sterile PBS solution to obtain a resuspended solution; the resuspended solution is filtered using a filter to obtain a deer antler polypeptide solution.

[0007] The composition of the osteogenic differentiation induction culture medium is as follows: Dexamethasone: 100 nM; L-Ascorbic acid: 50μM; β-glycerophosphate sodium: 10mM; Deer antler polypeptide solution: 200μg / mL~1000μg / mL; The complete culture medium was a DMEM:F12 = 1:1 medium containing 10% fetal bovine serum and 1% penicillin and streptomycin.

[0008] The diameter of the small pieces of deer antler in S1 is 0.2 to 0.5 cm.

[0009] The centrifugation conditions in S2 are centrifugation at 1000-5000 rpm for 10 minutes.

[0010] The particle size of the deer antler tissue powder in S3 is 100-500 μm.

[0011] The protease inhibitor added in S4 and S5 accounts for 0.01% to 0.05% of the total mass; the extraction conditions are 4 to 10°C for 12 to 48 hours.

[0012] The filter membrane used in S6 has a pore size of 0.45 μm; the dialysis bag used in S7 has a specification of 500 Da, and the dialysis treatment lasts for 12 hours.

[0013] The filter used in S9 has a pore size of 0.22 μm.

[0014] An osteogenic induction differentiation method, wherein mesenchymal stem cells are cultured using the above-mentioned osteogenic differentiation induction culture medium based on deer antler polypeptide.

[0015] The mesenchymal stem cells mentioned are human bone marrow mesenchymal stem cells or rat bone marrow mesenchymal stem cells.

[0016] The working process and working principle of this invention: This invention uses the above-mentioned deer antler polypeptide osteogenic differentiation induction medium to culture mesenchymal stem cells.

[0017] Mesenchymal stem cells are human bone marrow mesenchymal stem cells (h-BMSCs) or rat bone marrow mesenchymal stem cells (r-BMSCs). The deer antler polypeptide is obtained by cutting deer antler into pieces, removing blood, pulverizing to obtain powder, and then repeatedly extracting, filtering, and dialyzing the powder to obtain a purified solution. Finally, the purified solution is freeze-dried to obtain the deer antler polypeptide.

[0018] The beneficial effects of this invention are: The deer antler polypeptide described in this invention, when used as an additive in osteogenic induction culture medium, can significantly improve the osteogenic differentiation effect of mesenchymal stem cells, and is superior to commercial osteogenic induction culture medium in certain scenarios.

[0019] The osteogenic differentiation induction method described in this invention improves upon the shortcomings of existing technologies by using deer antler polypeptide osteogenic differentiation induction medium, which can enhance the osteogenic differentiation effect of mesenchymal stem cells. Attached Figure Description

[0020] Figure 1 The results show the KEGG pathway enrichment of the components contained in the deer antler polypeptide in the embodiments of the present invention.

[0021] Figure 2 This is a size exclusion chromatographic diagram of deer antler polypeptide in an embodiment of the present invention.

[0022] Figure 3 This is an SDS-page identification diagram of deer antler polypeptide in an embodiment of the present invention.

[0023] Figure 4 This is a comparison chart of the effects of different concentrations of deer antler polypeptide in the embodiments of the present invention.

[0024] Figure 5 This is a comparison of the ALP colorimetric effects of the osteogenic induction medium based on deer antler polypeptide and other osteogenic induction media in this embodiment of the invention.

[0025] Figure 6 This is a comparison of the ARS staining effects of the osteogenic induction medium based on deer antler polypeptide and other osteogenic induction media in this embodiment of the invention.

[0026] Figure 7 This is a comparison chart of the Runx-2 effect between the osteogenic induction culture medium based on deer antler polypeptide and other osteogenic induction culture media in this embodiment of the invention. Detailed Implementation

[0027] Please see Figures 1 to 7 The figures shown are embodiments and comparative examples of the present invention. Example 1

[0028] An osteogenic differentiation induction culture medium based on deer antler polypeptides contains deer antler polypeptide solution, dexamethasone, L-ascorbic acid, sodium β-glycerophosphate, and complete culture medium; wherein, Dexamethasone: 100 nM; L-Ascorbic acid: 50μM; β-glycerophosphate sodium: 10mM; Deer antler polypeptide solution: 200μg / mL~1000μg / mL; The complete culture medium was a DMEM:F12 = 1:1 medium containing 10% fetal bovine serum and 1% penicillin and streptomycin.

[0029] The deer antler polypeptide solution is prepared by the following steps: S1. Cut fresh two-pronged deer antlers and peel off the skin. Cut the antler tissue into pieces to obtain small pieces of antler with a diameter of 0.2cm. S2. Place the small pieces of deer antler obtained in S1 into an empty centrifuge column and centrifuge at 3000 rpm for 10 min to remove blood, and obtain small pieces of deblood-free deer antler tissue. S3. The small pieces of deer antler tissue after blood removal in S2 are pulverized to obtain deer antler tissue powder with a particle size of 300μm. S4. Mix the deer antler tissue powder obtained in S3 with water at a ratio of 1g:10mL, and add 0.02% serine protease inhibitor by mass to obtain a mixture; place the mixture in an environment at 4℃ for 24h for extraction, filter, and store the resulting extract separately. S5. Mix the solid deer antler tissue obtained from S4 with water at a ratio of 1g:10mL, and add 0.02% serine protease inhibitor by mass to obtain a mixture again. Place the mixture at 4℃ for 24h for extraction, filter, and store the resulting extract separately. Repeat this step twice. Mix all the above extracts to obtain a crude extract. S6. The crude extract obtained in S5 is filtered using a filter membrane with a pore size of 0.45 μm to obtain the filtrate. S7. Place the filtrate obtained in S6 into a dialysis bag with a specification of 500 Da and dialyze for 12 hours to remove inorganic salts and obtain a purified solution. S8. The purified solution obtained in S7 is freeze-dried to obtain a dried powder, which is the deer antler polypeptide extract. S9. The deer antler polypeptide extract obtained in S8 is resuspended and dissolved in sterile PBS solution to obtain a resuspended solution; then the resuspended solution is filtered using a filter with a pore size of 0.22 μm to obtain a deer antler polypeptide solution.

[0030] The preparation method of the deer antler polypeptide osteogenic induction culture medium in Example 1 is as follows: (1) Preparation of dexamethasone: Weigh an appropriate amount of dexamethasone, dissolve it in anhydrous ethanol to prepare a 1mM stock solution, filter it through a 0.22μm filter membrane for sterilization, dispense it into EP tubes, and store it at -20℃.

[0031] (2) Preparation of L-ascorbic acid: Weigh L-ascorbic acid, dissolve it in PBS buffer to prepare a 50mM stock solution, filter it through a 0.22μm filter membrane for sterilization, dispense it into EP tubes, and store it at -20℃.

[0032] (3) Preparation of sodium β-glycerophosphate: Weigh an appropriate amount of sodium β-glycerophosphate, dissolve it in PBS buffer to prepare a 1M stock solution, filter it through a 0.22μm filter membrane for sterilization, dispense it into EP tubes, and store it at -20℃.

[0033] (4) Preparation of osteogenic induction medium: In DMEM:F12=1:1 medium, take the corresponding amounts of fetal bovine serum, penicillin and streptomycin, dexamethasone, L-ascorbic acid, sodium β-glycerophosphate and deer antler polypeptide according to the complete medium of Example 1, and then mix them evenly to obtain the required osteogenic induction medium. Example 2

[0034] An osteogenic induction differentiation method is provided, wherein human bone marrow mesenchymal stem cells and rat bone marrow mesenchymal stem cells are cultured using the osteogenic differentiation induction culture medium based on deer antler polypeptide described in Example 1.

[0035] Comparative Example 1 Human bone marrow mesenchymal stem cells and rat bone marrow mesenchymal stem cells were cultured using an osteogenic induction medium. The osteogenic induction medium had the same characteristics as in Example 1, except that the raw material content was: deer antler polypeptide: 0 μg / mL.

[0036] The preparation method of the osteogenic induction culture medium for deer antler polypeptide in Comparative Example 1 is as follows: (1) Preparation of dexamethasone: Weigh an appropriate amount of dexamethasone, dissolve it in anhydrous ethanol to prepare a 1mM stock solution, filter it through a 0.22μm filter membrane for sterilization, dispense it into EP tubes, and store it at -20℃.

[0037] (2) Preparation of L-ascorbic acid: Weigh L-ascorbic acid, dissolve it in PBS buffer to prepare a 50mM stock solution, filter it through a 0.22μm filter membrane for sterilization, dispense it into EP tubes, and store it at -20℃.

[0038] (3) Preparation of sodium β-glycerophosphate: Weigh an appropriate amount of sodium β-glycerophosphate, dissolve it in PBS buffer to prepare a 1M stock solution, filter it through a 0.22μm filter membrane for sterilization, dispense it into EP tubes, and store it at -20℃.

[0039] (4) Preparation of osteogenic induction medium: In DMEM:F12=1:1 medium, take the corresponding amounts of fetal bovine serum, penicillin and streptomycin, dexamethasone, L-ascorbic acid and β-glycerophosphate sodium according to the complete medium of this embodiment, and then mix them evenly to obtain the required osteogenic induction medium.

[0040] Comparative Example 2 Human bone marrow mesenchymal stem cells and rat bone marrow mesenchymal stem cells were cultured using Xirui Infinite (Hangzhou) mesenchymal stem cell osteogenic induction differentiation medium.

[0041] Comparative Example 3 Human bone marrow mesenchymal stem cells and rat bone marrow mesenchymal stem cells were cultured separately using Pronosai (Wuhan) mesenchymal stem cell osteogenic differentiation induction medium.

[0042] in conclusion Performance testing and results analysis 1. Proteomics analysis Please see Figure 1 As shown, deer antler polypeptide samples were extracted and fully dissolved, followed by enzymatic hydrolysis. The resulting polypeptides were desalted and purified. Subsequently, the polypeptide mixture was analyzed using a nanoElute ultra-high performance liquid chromatography system (Bruker, Germany). Mass spectrometry was performed on a timsTOF-Pro2 mass spectrometer in data-independent acquisition (DIA) mode to generate raw spectral data. The acquired DIA data were compared with protein databases using Spectronaut software (Biognosys, Switzerland) to identify polypeptides and proteins. KEGG pathway analysis was then performed.

[0043] 2. Molecular weight distribution of deer antler polypeptide solution Please see Figure 2 As shown, size exclusion chromatography (SEC) analysis of deer antler polypeptide (VAP) Molecular weight distribution was analyzed using size exclusion chromatography (SEC). The mobile phase was 150 mmol phosphate buffer. An SRT-CSEC-300 column (7.8 × 300 mm, 5 μm particle size) was used, and the main instrument was a high-performance liquid chromatography system equipped with a UV detector. For sample preparation, an appropriate amount of test sample was diluted to a concentration of 1 μg / μL and then centrifuged at 12,000 rpm for 10 min at 4 °C. The supernatant was collected as the analytical sample. The chromatographic conditions were set as follows: column temperature 25 °C, flow rate 0.7 mL / min, injection volume 20 μL, and detection wavelength 280 nm. At least three suitable standard references were selected, and molecular weight calibration was performed based on the actual detection results. Finally, the elution chromatogram of the sample was obtained, and the relative molecular weight and molecular weight distribution parameters were calculated using the calibration curve of the standard references.

[0044] Please see Figure 3 As shown, the sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of deer antler peptides is presented. The molecular weight of the samples was analyzed by SDS-PAGE. In short, the samples were diluted to a concentration of 1 μg / μL and mixed with an equal volume of 2×SDS loading buffer, followed by denaturation at 95°C for 5 min. The proteins were then added to a 12% polyacrylamide gel and electrophoresed at a constant voltage of 120 V until the dye front reached the bottom of the gel. A pre-stained protein molecular weight standard reference was run simultaneously to calibrate the sample molecular weight. After electrophoresis, the samples were stained with Coomassie Brilliant Blue R-250 for 1 h, followed by destaining until clear protein bands appeared. The molecular weight of each protein was determined by comparing its migration distance to that of the corresponding standard reference, and the relevant molecular weight distribution parameters were calculated.

[0045] 3. Osteogenesis induction effect detection (1) Cell seeding: In a 24-well plate, seed 5 × 10⁶ cells per well. 4 A number of human bone marrow mesenchymal stem cells (h-BMSCs) and rat bone marrow mesenchymal stem cells (r-BMSCs) were cultured. After the cells adhered and grew for 24 hours, they were cultured in the corresponding osteogenic induction medium at 37°C and 5% CO2. The medium was changed every 48 hours.

[0046] (2) Alkaline phosphatase (ALP) detection: On day 6 of osteogenic induction, alkaline phosphatase expression was detected using an alkaline phosphatase colorimetric kit (Beyotime, China).

[0047] (3) Alizarin Red (ARS) staining: On day 12 of osteogenic induction, the formation of calcium nodules was detected using Alizarin Red staining solution (Cyagen Biosciences, China).

[0048] (4) Immunofluorescence staining of Runx-2 protein: Immunofluorescence staining of Runx-2 protein was performed 6 days after induction to assess the expression of Runx-2 protein. After fixation, permeabilization and blocking, the cells were incubated overnight with the primary antibody of Runx-2 protein (Aibotek, China), followed by incubation with the secondary antibody and counterstaining of the nuclei with DAPI (4',6-diamidindo-2-phenylindole). The samples were photographed and observed using a fluorescence microscope.

[0049] 4. Determination of the optimal concentration of deer antler polypeptide in osteogenic induction culture medium Please see Figure 4 As shown, cells were seeded according to the above method, and then cultured in osteogenic induction medium containing different concentrations of deer antler polypeptide (200 μg / mL, 400 μg / mL, 600 μg / mL, 800 μg / mL, 1000 μg / mL) at 37℃ and 5% CO2. The medium was changed every 48 hours. ALP was detected on day 6 of osteogenic induction, and ARS staining was performed on day 12. The results showed that when the concentration of deer antler polypeptide was 600-800 μg / mL, the ALP staining was more intense, and more red areas of calcium nodules were shown in ARS staining. The optimal concentration of deer antler polypeptide for osteogenic induction was 600-800 μg / mL.

[0050] 5. Comparison of the osteogenic induction effects of deer antler polypeptide osteogenic induction medium and comparative medium Cells were seeded as described above and then cultured in different osteogenic induction media (Example 2, Comparative Example 1, Comparative Example 2, Comparative Example 3) at 37°C and 5% CO2. The medium was changed every 48 hours. ALP detection and Runx-2 immunofluorescence staining were performed on day 6 of osteogenic induction, and ARS staining was performed on day 12. The results showed that ALP staining was more intense in Example 2 compared to Comparative Examples 1, 2, and 3 (see [link to relevant documentation]). Figure 5 Compared to Comparative Examples 1 and 2, ARS staining in Example 2 showed more calcium nodules, similar to Comparative Example 3 (see Comparative Example 3). Figure 6 Compared to Comparative Examples 1, 2, and 3, Runx-2 protein immunofluorescence staining in Example 2 showed an overall higher red fluorescence intensity (see [link to example]). Figure 7 ); Figure 7 In the text, Runx-2 is explained as Runt-related transcription factor 2; Merge: the image is a combination of the images corresponding to Runx-2 and DAPI, superimposed together.

[0051] The results showed that the deer antler polypeptide osteogenic medium exhibited excellent ability to promote the osteogenic differentiation of BMSCs, not only significantly improving the osteogenic differentiation effect, but also outperforming the induction effect of commercially available osteogenic induction medium products.

Claims

1. A polypeptide based on pilose antler for inducing osteogenic differentiation of a culture medium, characterized by: The polypeptide solution of pilose antler, dexamethasone, L-ascorbic acid, beta-glycerophosphate sodium and complete medium; The polypeptide solution of pilose antler is prepared by the following steps: S1, cutting fresh two horn pilose antler and peeling off the skin, cutting the pilose antler tissue into small pieces, obtaining pilose antler small pieces; S2, placing the pilose antler small pieces obtained in S1 in a centrifugal empty column tube, centrifuging to remove blood, obtaining pilose antler tissue small pieces after blood removal; S3, crushing the pilose antler tissue small pieces after blood removal in S2 to obtain pilose antler tissue powder; S4, mixing the pilose antler tissue powder obtained in S3 with water at a ratio of 1g:10-20mL, and adding protease inhibitors, obtaining a mixed solution; the mixed solution is extracted, filtered, and the obtained extract is stored separately; S5, mixing the solid pilose antler tissue obtained by filtering in S4 with water at a ratio of 1g:10-20mL, and adding protease inhibitors, obtaining a mixed solution again, extracting the mixed solution, filtering, and storing the obtained extract; repeat this step several times; mix all the above extracts to obtain a crude extract; S6, using a filter membrane to perform suction filtration on the crude extract obtained in S5 to obtain a filtrate; S7, dialysis treatment of the filtrate obtained in S6 to remove inorganic salts to obtain a purified solution; S8, freeze-drying treatment of the purified solution obtained in S7, and the obtained dry powder is pilose antler polypeptide extract; S9, resuspending and dissolving the pilose antler polypeptide extract obtained in S8 with sterile PBS solution to obtain a resuspended solution; Filtering the resuspended solution using a filter to obtain a pilose antler polypeptide solution.

2. The osteogenic differentiation induction culture medium based on deer antler polypeptide according to claim 1, characterized in that: The content of each component is as follows: Dexamethasone: 100nM; L-ascorbic acid: 50μM; Beta-glycerophosphate sodium: 10mM; Pilose antler polypeptide solution: 200μg / mL-1000μg / mL; The complete medium is a DMEM:F12=1:1 medium containing 10% fetal bovine serum and 1% penicillin-streptomycin.

3. The osteogenic differentiation induction culture medium based on deer antler polypeptide according to claim 1, characterized in that: The diameter of the pilose antler small pieces in S1 is 0.2-0.5cm.

4. The polypeptide-based osteogenic differentiation inducing medium according to claim 1, wherein the polypeptide is a polypeptide derived from Cervus elaphus.

5. The centrifugation conditions in S2 are 1000-5000rpm for 10min.

5. The polypeptide-based osteogenic differentiation inducing medium according to claim 1, wherein the polypeptide is a polypeptide derived from Cervus elaphus.

6. The polypeptide-based osteogenic differentiation inducing medium according to claim 1, wherein the polypeptide is a polypeptide derived from Cervus nippon. The particle size of the pilose antler tissue powder in S3 is 100-500μm.

6. The polypeptide-based osteogenic differentiation inducing medium according to claim 1, wherein the polypeptide is a polypeptide derived from a Cervus elaphus Linnaeus. 6 The mass ratio of the protease inhibitor added in S4 and S5 is 0.01%-0.05%; the extraction conditions are 4-10℃ for 12-48h.

7. The polypeptide-based osteogenic differentiation inducing medium according to claim 1, wherein the polypeptide is a polypeptide derived from Cervus elaphus.

8. The polypeptide-based osteogenic differentiation inducing medium according to claim 1, wherein the polypeptide is a polypeptide derived from Cervus nippon. The pore size of the filter membrane used in S6 is 0.45μm; the dialysis bag used in S7 is 500Da, and the dialysis treatment is 12h.

8. The polypeptide-based osteogenic differentiation inducing medium according to claim 1, wherein the polypeptide is a polypeptide derived from a Cervus elaphus. The pore size of the filter used in S9 is 0.22μm. ​ 9. A method for inducing differentiation into osteogenesis, characterized by: The mesenchymal stem cells are cultured using the pilose antler polypeptide-based osteogenic differentiation induction medium described in any one of claims 1-8.

10. The method of osteogenic induction and differentiation according to claim 9, wherein: The mesenchymal stem cells are human bone marrow mesenchymal stem cells or rat bone marrow mesenchymal stem cells.

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