Application of Oroxylum indicum glycoside B in the preparation of drugs that promote osteogenic differentiation of bone marrow mesenchymal stem cells, its hydrogel and preparation method

Abstract

This invention discloses the application of oroxylin and oleracea oleifera glycoside B in the preparation of drugs that promote osteogenic differentiation of bone marrow mesenchymal stem cells, its hydrogel, and preparation method, belonging to the field of biomedical materials technology. Firstly, the application of oroxylin and oleifera glycoside B in the preparation of drugs that promote osteogenic differentiation of bone marrow mesenchymal stem cells is provided. A self-assembled polypeptide hydrogel loaded with oroxylin and oleifera glycoside B is also provided. The hydrogel is obtained by combining oroxylin and oleifera glycoside B with a self-assembled polypeptide of a specific sequence (SEQ ID No. 1) through mild physical blending and incubation. This hydrogel forms a three-dimensional nanofiber network that can in situ encapsulate and continuously release oroxylin and oleifera glycoside B. It can not only effectively recruit bone marrow mesenchymal stem cells but also provide an ideal microenvironment for their proliferation and osteogenic differentiation, significantly upregulating the expression of osteogenic-related genes, thereby potentially accelerating bone defect repair.

Description

Technical Field

[0001] This invention belongs to the field of biomedical materials technology, specifically the application of oroxylum inophylline B in the preparation of drugs that promote osteogenic differentiation of bone marrow mesenchymal stem cells, its hydrogel, and its preparation method. Background Technology

[0002] Bone defects are a common clinical challenge caused by factors such as trauma, infection, tumor resection, and metabolic bone diseases. Their repair outcomes directly impact patients' limb function and quality of life. Unlike soft tissue injuries, bone tissue possesses a certain degree of self-healing ability; however, when the defect volume exceeds a critical-size defect, the body cannot achieve spontaneous healing, and the remaining bone cavity is easily occupied by fibrous connective tissue, leading to delayed healing or even nonunion. Currently, autologous bone grafting is still considered the "gold standard" for bone defect repair, but its widespread application is significantly limited by drawbacks such as limited donor bone volume, donor site complications, poor donor-recipient matching, and secondary surgical trauma.

[0003] Bone marrow mesenchymal stem cells (BMSCs) play a crucial regulatory role in the natural repair of bone defects. In the early stages of injury, BMSCs are recruited to the defect area under the guidance of inflammatory and chemokine gradients. Subsequently, they regulate macrophage polarization through paracrine mechanisms, inhibiting excessive inflammation. In the mid-repair phase, BMSCs proliferate and differentiate into osteogenic progenitor cells, secreting key factors such as bone morphogenetic protein-2 (BMP-2) and vascular endothelial growth factor (VEGF). These factors both induce osteogenic differentiation and promote angiogenesis, laying the biological and mechanical foundation for subsequent mineralization. Finally, during the remodeling phase, BMSCs regulate bone matrix remodeling through matrix metalloproteinases (MMPs), achieving lamellar bone replacement and Haver system remodeling. However, large bone defects often result in local microenvironment ischemia, persistent inflammation, accumulation of reactive oxygen species (ROS), and insufficient mechanical stability, leading to low recruitment efficiency, poor survival, and incomplete osteogenic differentiation of endogenous BMSCs, ultimately resulting in "arrested bone healing." Therefore, how to efficiently recruit and precisely regulate the osteogenic differentiation of bone marrow mesenchymal stem cells has become a key scientific issue in overcoming the bottleneck of regeneration of large bone defects.

[0004] Oroxylum indicum glycoside B is a flavonoid glycoside extracted from the traditional Chinese medicine Oroxylum indicum. Studies have shown that it possesses certain anti-inflammatory and antioxidant bioactivities. Self-assembled peptide hydrogels, due to their excellent biocompatibility, injectability, and ability to mimic the extracellular matrix (ECM) nanofiber structure, have demonstrated great potential in tissue engineering. Currently, there are no reports on combining Oroxylum indicum glycoside B with specific self-assembled peptides to construct injectable hydrogel systems for actively regulating the behavior of bone marrow mesenchymal stem cells and promoting bone defect repair. Summary of the Invention

[0005] The purpose of this invention is to overcome the shortcomings of the prior art, provide a new application of oroxylin and oxyphylla glycoside B in promoting osteogenic differentiation of bone marrow mesenchymal stem cells, and further provide an injectable self-assembled polypeptide hydrogel that can efficiently load and sustainably release oroxylin and oxyphylla glycoside B. This hydrogel can simulate the bone tissue microenvironment in vivo, effectively promote the recruitment, proliferation and osteogenic differentiation of bone marrow mesenchymal stem cells, thereby accelerating the repair of bone defects.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] In a first aspect, the present invention provides the use of oroxylon ammodendronin B or a pharmaceutically acceptable salt thereof in the preparation of a medicament for promoting osteogenic differentiation of bone marrow mesenchymal stem cells.

[0008] Preferably, the drug can also promote the proliferation and osteogenic differentiation of bone marrow mesenchymal stem cells.

[0009] Oroxin B (English name: Oroxin B) is derived from the Bignoniaceae plant Orochi (Orochi spp.) Oroxylum indicum A flavonoid glycoside compound was extracted and isolated from [a specific source]. Its CAS number is 114482-86-9, and its chemical structural formula is as follows:

[0010]

[0011] Secondly, the present invention provides a hydrogel of a self-assembled polypeptide loaded with oroxylin B. The hydrogel comprises oroxylin B and a self-assembled polypeptide, wherein the amino acid sequence of the self-assembled polypeptide is shown in SEQ ID No. 1 (SKPPGTSSFFVLE).

[0012] Preferably, the oroxylon ammodendronin B is in situ loaded in the three-dimensional nanofiber network structure formed by the self-assembly of the self-assembled polypeptide, and can be continuously released from the structure.

[0013] Preferably, the hydrogel can mimic the in vivo bone tissue microenvironment, promoting the recruitment, adhesion, and proliferation of bone marrow mesenchymal stem cells.

[0014] Preferably, the hydrogel can upregulate the expression of osteogenic-related genes (such as Runx2, OPN, BMP-2, etc.) in bone marrow mesenchymal stem cells, inducing them to differentiate into osteoblasts.

[0015] Thirdly, the present invention provides a method for preparing the above-mentioned self-assembled polypeptide hydrogel loaded with oroxylin B, characterized by comprising the following steps:

[0016] (1) Disperse oroxylin B in phosphate buffer (PBS), and dissolve by sonication in the dark to obtain working solution of oroxylin B;

[0017] (2) Add the self-assembled peptide to the working solution, shake to mix, and obtain a mixture;

[0018] (3) The mixture is incubated at 30-40°C in the dark with shaking for 8-24 hours to obtain the hydrogel.

[0019] Preferably, in step (1), the pH of the phosphate buffer is 7.2-7.4, and the concentration of oroxylum indicum B is 2.5-100 μM, more preferably 40 μM.

[0020] Preferably, in step (2), the final concentration of the self-assembled polypeptide is 0.02-0.05 g / mL, more preferably 0.03 g / mL.

[0021] Preferably, the oscillation incubation temperature in step 3 is 37°C.

[0022] Fourthly, the present invention provides the application of the above-mentioned self-assembled polypeptide hydrogel loaded with oroxylin B in the preparation of biomaterials for repairing bone defects.

[0023] Preferably, the medical material is an injectable bone repair material.

[0024] Fifthly, the present invention also provides the application of the above-mentioned self-assembled polypeptide hydrogel loaded with oroxylin B in the preparation of biomaterials for promoting osteogenic differentiation of bone marrow mesenchymal stem cells.

[0025] The biomaterial can promote the recruitment and proliferation of bone marrow mesenchymal stem cells, upregulate the expression of osteogenic-related genes (such as Runx2, OPN, BMP-2, etc.) of bone marrow mesenchymal stem cells, and induce them to differentiate into osteoblasts.

[0026] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0027] This invention reveals for the first time the clear use of oroxylon ammodendronin B in promoting osteogenic differentiation of bone marrow mesenchymal stem cells, providing a new candidate drug molecule for bone regeneration therapy.

[0028] An injectable hydrogel was constructed by combining oroxylin and oleracea glycoside B with a self-assembling polypeptide of a specific sequence. This carrier can continuously and controllably release oroxylin and oleracea glycoside B, precisely regulating the behavior of bone marrow mesenchymal stem cells in time and space.

[0029] This hydrogel system can effectively mimic the bone tissue microenvironment, significantly promote the chemotactic migration (recruitment) and proliferation of bone marrow mesenchymal stem cells in vitro, and upregulate the expression of osteogenic-related genes, inducing their differentiation into osteoblasts and the formation of mineralized nodules.

[0030] The preparation process of hydrogels is carried out under mild conditions (physiological temperature and pH), without the need to add any chemical cross-linking agents or organic solvents, thus avoiding potential toxicity, ensuring high biosafety, and facilitating immediate clinical use.

[0031] This injectable hydrogel can be minimally invasively implanted into bone defect sites, making it suitable for filling irregular defects and providing a novel biomaterial platform for the regenerative treatment of bone defects that is efficient, safe, and convenient. Attached Figure Description

[0032] Figure 1 The results of CCK-8 assay used in Example 1 to detect the cell viability of bone marrow mesenchymal stem cells after 48 hours of treatment with different concentrations of oroxylum indicum B.

[0033] Figure 2 This is a grayscale scan of Runx2, Opn, Bmp2, and β-actin in bone marrow mesenchymal stem cells of the control group and the oroxylin B group in Example 2.

[0034] Figure 3 Visual representations of HG (hydrogel) and HG@OB (hydrogel loaded with oroxylin B) prepared in Example 3;

[0035] Figure 4 Scanning electron microscope images of HG and HG@OB prepared in Example 3;

[0036] Figure 5 The rheological storage modulus / loss modulus curves of HG and HG@OB prepared in Example 3;

[0037] Figure 6 The sustained-release curves of HG and HG@OB prepared in Example 3;

[0038] Figure 7 The results of CCK-8 assays on the cell viability of bone marrow mesenchymal stem cells after 48 h of treatment with different drug concentrations of HG@OB in Example 7.

[0039] Figure 8 These are Calcein / PI double-stained fluorescence images of bone marrow mesenchymal stem cells at different drug concentrations of HG@OB in Example 8, showing cell viability and cytotoxicity.

[0040] Figure 9 The blank control in Example 9 is an image of Transwell crystal violet staining of bone marrow mesenchymal stem cells by HG and HG@OB.

[0041] Figure 10 HG was added to the bone marrow mesenchymal stem cells in Example 10. After culturing HG@OB for 5 days, the early osteogenic induction map was detected by BCIP / NBT alkaline phosphatase colorimetric assay.

[0042] Figure 11 In Example 10, bone marrow mesenchymal stem cells were added with HG and cultured at HG@OB for 14 days. Osteogenic mineralization was detected by Alizarin Red S staining. Detailed Implementation

[0043] The present invention will now be described in further detail with reference to the accompanying drawings and embodiments, but this does not limit the present invention in any way. Any modifications or improvements made based on the teachings of the present invention shall fall within the protection scope of the present invention.

[0044] The processes, conditions, reagents, and experimental methods used in implementing this invention, except as specifically mentioned below, are all common knowledge and general knowledge in the field, and this invention does not have any particular limitations. Experimental methods in the embodiments that do not specify specific conditions are generally performed under conventional conditions or as recommended by the manufacturer.

[0045] Unless otherwise stated, all technical terms and scientific terms used in this specification have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. However, in the event of any conflict, the specification containing the definitions shall prevail.

[0046] The following is information on the main reagent sources in the examples:

[0047] The peptide Ser-Lys-Pro-Pro-Gly-Thr-Ser-Ser-Phe-Phe-Val-Leu-Glu was purchased from Jier Biochemical (Shanghai) Co., Ltd.; Oroxylum indicum B was purchased from Chengdu Yirui Biotechnology Co., Ltd.; the CCK-8 cell proliferation / toxicity assay kit was purchased from TargetMol Chemicals Inc. (Boston, USA); the Calcein / PI double staining cell viability and cytotoxicity assay kit and the BCIP / NBT alkaline phosphatase colorimetric kit were both purchased from Shanghai Beyotime Biotechnology Co., Ltd.; Alizarin Red S staining solution was purchased from Suzhou Haixing Biotechnology Co., Ltd.; the crystal violet staining solution was purchased from Beijing Solarbio Science & Technology Co., Ltd.; Runx2, Opn and β-actin antibodies were all purchased from Wuhan Sanying Biotechnology Co., Ltd.; α-MEM medium, fetal bovine serum (FBS), penicillin-streptomycin mixed antibiotic and trypsin-EDTA digestion solution were all purchased from Eva Biopharmaceutical Technology Co., Ltd. (Suzhou, China); phosphate buffered saline (PBS, pH 7.4) was purchased from Savill Biotechnology Co., Ltd. (Wuhan, China).

[0048] Example 1

[0049] Bone marrow mesenchymal stem cells were used at a rate of 5 × 10⁻⁶. 3Cells were seeded at various densities in 96-well plates. After overnight adhesion, different concentrations of oroxylon ammodendronin B were added. After 48 hours of culture, the plates were removed, the culture medium was aspirated, and the cells were washed twice with PBS. CCK-8 stock solution and culture medium were mixed at a 1:9 ratio to prepare CCK-8 working solution. 130 μL of CCK-8 working solution was added to each well in the dark and incubated for 1 hour. 100 μL of the solution was aspirated into new 96-well plates, with three sub-wells for each sample. The absorbance (OD value) of each plate at 450 nm was measured using a microplate reader to indirectly reflect cell viability.

[0050] The results are as follows Figure 1 As shown, within the low concentration range (0-5 μM), oroxylin and oleracea oleifera glycoside B promoted the proliferation of bone marrow mesenchymal stem cells (BMSCs), exhibiting a concentration-dependent increasing trend. The 5 μM concentration showed the most significant proliferative effect, with cell viability significantly improved compared to the control group (0 μM), suggesting that at this concentration, oroxylin and oleifera glycoside B can effectively activate the proliferative activity of BMSCs and has potential application value in scenarios requiring stem cell proliferation, such as bone tissue repair. When the concentration of oroxylin and oleifera glycoside B increased to 10 μM and above, it showed a significant inhibitory effect on BMSC proliferation, which increased with increasing concentration. This result indicates that high concentrations of oroxylin and oleifera glycoside B have significant cytotoxic effects on BMSCs, further verifying the bidirectional regulatory effect of oroxylin and oleifera glycoside B on BMSC proliferation.

[0051] Example 2

[0052] Bone marrow mesenchymal stem cells were used at a rate of 1×10 5 The cells were seeded in 6-well plates and cultured in an incubator at 37°C and 5% CO2 to induce osteogenic differentiation. Osteogenic induction medium was prepared, consisting of 50 μg / mL vitamin C, 10 mM sodium β-glycerophosphate, 100 nM dexamethasone, 10% FBS, and 1% P / S in α-MEM medium.

[0053] The control group received only osteogenic induction medium, while the oroxylin B group (OB) was induced with both osteogenic induction medium and oroxylin B (final concentration 5 μM). The cell culture medium was changed halfway every 2 days. After 5 days of induction, the culture medium was aspirated, the cells were washed twice with PBS, and an appropriate amount of high-efficiency RIPA cell lysis buffer (containing 1% PMSF) was added. The cells were lysed on ice for 30 min, collected with a cell scraper into 1.5 mL EP tubes, placed with grinding beads, and ground in a low-temperature homogenizer for 30 s. The cells were then centrifuged at 12,000 rpm at 4°C for 30 min. The protein supernatant was collected, and after protein quantification, 25 μg of total protein from each sample was added to a 7.5% SDS-PAGE lane for electrophoresis separation. The target protein was then transferred onto a PVDF membrane. After washing, antibodies Runx2 (1:2000), Opn (1:1000), Bmp2 (1:1000), and β-action (1:4000) were added, and the membrane was incubated overnight at 4°C. After washing with TBST three times for 10 minutes, the membrane was incubated with the corresponding secondary antibody at room temperature for 2 hours. After washing, chemiluminescent reagents were added, and the membrane was exposed and developed. The grayscale values ​​were scanned using ImageJ software.

[0054] The results are as follows Figure 2 As shown, Western blotting results confirmed that the expression levels of Bmp2, Opn, and Runx2 were higher in the oroxylin B group, indicating that oroxylin B can promote osteogenic differentiation of bone marrow mesenchymal stem cells.

[0055] Example 3

[0056] This embodiment provides a method for preparing a self-assembled polypeptide hydrogel loaded with oroxylon ammodendronin B, the specific steps of which are as follows:

[0057] Different masses of oroxylum indicum B powder were dispersed in phosphate-buffered saline (PBS) to prepare oroxylum indicum B dispersions of different concentrations (2.5, 5, 10, 20, 40, 80, 100 μM). The dispersions were then dissolved by sonication in the dark for 10 min at 37°C, and the resulting dispersions were used as working solutions. A self-assembled peptide (Ser-Lys-Pro-Pro-Gly-Thr-Ser-Ser-Phe-Phe-Val-Leu-Glu, i.e., SKPPGTSSFFVLE) was added to the working solution at a concentration of 0.03 g / mL and thoroughly mixed by shaking on a horizontal shaker for 10 min. The samples were then incubated at 37°C with gentle shaking in the dark to obtain drug-loaded hydrogels of different concentrations (2.5, 5, 10, 20, 40, 80, 100 μM), denoted as HG@OB.

[0058] Alternatively, without adding oroxylin B, a self-assembled polypeptide hydrogel without oroxylin B was prepared according to the above method, denoted as HG.

[0059] like Figure 3 As shown, HG and HG@OB (drug loading concentration 100 μM) prepared by visual observation are both translucent gel structures encapsulated in a pointed-bottom container; their overall morphology is similar, and no obvious structural defects such as collapse or cracking are observed; both hydrogels are stably filled in the container, and the gel structure at the bottom is intact, indicating that both have good formability and structural stability.

[0060] Example 4

[0061] The blank hydrogel and the hydrogel loaded with phalloidin B (drug loading concentration 100 μM) prepared in Example 3 were pre-frozen at -80°C for 24 h, then transferred to a freeze dryer and freeze-dried for 48 h to obtain a dried porous scaffold. The freeze-dried scaffold was fixed to the SEM sample stage with conductive adhesive and its surface was sputter-coated with gold. The dried scaffold with the gold-coated carbon substrate (Hides Gold on Carbon) was quickly placed in the sample chamber of a Thermo Scientific field emission scanning electron microscope. Under high vacuum mode, the chamber pressure was confirmed to be stable at 5.4–7.6 × 10⁻⁶. -4 After Pa, the accelerating voltage was set to 1.5 kV, the working distance to 9.2–9.3 mm, and imaging was performed using a secondary electron (SE) signal and an Everhart-Thornley detector (ETD). Images were acquired at random magnifications of 2000×, 5000×, and 10000× to compare the internal pore structure of the hydrogel and the effect of oroxylin B loading on the pore size distribution.

[0062] Scanning electron microscopy (SEM) images of the blank hydrogel (HG) and the hydrogel loaded with oroxylin B (HG@OB) are shown below. Figure 4 As shown in the electron micrographs, the characteristics of the two groups of samples are as follows: The HG group exhibits a three-dimensional porous network structure with uneven pore sizes (ranging from tens of micrometers to several micrometers) and relatively smooth pore wall surfaces; as the magnification increases (from 2000×→5000×→10000×), the pore wall details become clearer, and there is no obvious exogenous particle attachment. The HG@OB group retains the porous network of HG, but a large number of fine particles (i.e., the loaded orbicularisoside B) are attached to the pore wall surface; after magnification, the particle distribution is relatively uniform, covering the pore wall surface, without disrupting the overall porous structure of the hydrogel.

[0063] Example 5

[0064] Cylindrical blank hydrogel (HG) and hydrogel sample loaded with oroxylin B (drug loading concentration 100 μM) (HG@OB) (Φ 20 mm × 2 mm) (prepared in Example 3) were tested using a rotational rheometer (flat plate fixture). The sample was placed on a parallel plate fixture, the Peltier temperature control was set to 25°C, the fixture gap matched the sample thickness (2 mm), and deionized water was dropped on the edge of the sample to form a water ring to prevent water evaporation. Selecting the dynamic oscillation mode, we first determined the 1% strain as the linear viscoelastic region using strain scanning, and then performed a dynamic frequency scan: the scanned frequencies were 0.01 Hz, 0.0126 Hz, 0.0158 Hz, 0.02 Hz, 0.0251 Hz, 0.0316 Hz, 0.0398 Hz, 0.0501 Hz, 0.0631 Hz, 0.0794 Hz, 0.1 Hz, 0.126 Hz, 0.158 Hz, 0.2 Hz, 0.251 Hz, 0.316 Hz, 0.398 Hz, 0.501 Hz, 0.631 Hz, 0.794 Hz, 1 Hz, 1.26 Hz, 1.58 Hz, 2 Hz, 2.51 Hz, 3.16 Hz, 3.98 Hz, 5.01 Hz, and 6.31 Hz. Record the curves of energy storage modulus (G′) and loss modulus (G″) as a function of frequency at Hz, 7.94 Hz, and 10 Hz.

[0065] Test results are as follows Figure 5 As shown, G′ and G″ of HG@OB are significantly higher than those of HG, and show a clear upward trend with increasing frequency in the 0.01–10 Hz range, indicating that loading of oroxylon ammodendronin B can effectively enhance the mechanical properties and viscoelastic response of the hydrogel. At the same time, G′ is always higher than G″, indicating that HG@OB is an "elasticity-dominated" viscoelastic material.

[0066] Example 6

[0067] A certain amount of hydrogel loaded with oroxylum indicum B (drug loading concentration 100 μM) (prepared in Example 3) was weighed and placed in 50 mL of phosphate-buffered saline (PBS, pH 7.4, 0.01 M). Release experiments were conducted at 37°C, protected from light, and under constant temperature shaking at 100 rpm. At 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10 days, 1 mL of release medium was accurately pipetted, and an equal volume of fresh PBS at the same temperature was added to maintain a constant total volume. The collected samples were immediately transferred to EP tubes and stored at -20°C protected from light until analysis. After all samples were collected, they were thawed at room temperature, and 10 μL was injected. The drug concentration was determined by HPLC, and release curves were plotted to evaluate the sustained-release performance of the drug in the hydrogel.

[0068] like Figure 6As shown in the figure, the drug release kinetic curve of the drug-containing hydrogel of the present invention demonstrates that the hydrogel has excellent sustained-release properties. In the initial release phase (0–2 days), oroxylin and oleracea oleifera glycoside B exhibits a mild burst release effect, with the concentration rapidly increasing to approximately 15 μM, quickly reaching an effective therapeutic concentration. In the middle phase (2–7 days), oroxylin and oleifera glycoside B enters a stable sustained release phase, with the concentration steadily increasing from 15 μM to approximately 35 μM, and the release rate is uniform, avoiding the toxic side effects caused by burst release of oroxylin and oleifera glycoside B. In the later phase (7–10 days), the release of oroxylin and oleifera glycoside B still maintains a stable increasing trend, with the concentration approaching 40 μM, and no obvious release plateau phase appears. This indicates that the hydrogel can achieve long-term and stable drug delivery, and has significant advantages in the treatment of chronic diseases or long-term local drug administration scenarios.

[0069] Example 7

[0070] The procedure was essentially the same as in Example 1, except that the drug treatment group was modified to use "in-situ hydrogel formation post-treatment": different concentrations (0, 2.5, 5, 10, 20, 40, 80, 100 μM) of oroxylin and oleoresin B were mixed with a polypeptide (final concentration 0.03 g / mL), filtered to remove bacteria, and this sterile mixture was added to a cell culture plate (40 μL per well in a 96-well plate). The plate was incubated at 37°C to allow the hydrogel to form in situ in the wells. After the hydrogel had stabilized, the cell suspension was gently inoculated onto the surface of the hydrogel, allowing oroxylin and oleoresin B to act on the cells through the sustained release of the gel.

[0071] The results are as follows Figure 7 As shown, bone marrow mesenchymal stem cells (BMSCs) were seeded onto hydrogels loaded with different concentrations of oroxylum indicum B. After 48 hours of culture, the effect on BMSC proliferation was evaluated using a CCK-8 assay. Within the concentration range of 0-40 μM, HG@OB exhibited a mild promoting effect on BMSC proliferation, with cell viability increasing with increasing concentration. The 40 μM concentration group showed the most significant proliferative effect, indicating that the hydrogel in this concentration range has good cell compatibility and proliferation-regulating activity. When the concentration increased above 40 μM, HG@OB began to inhibit BMSC proliferation in a concentration-dependent manner, but remained at a high level, indicating that even at high concentrations, the hydrogel still exhibited good cell safety and did not show severe cytotoxicity.

[0072] Those skilled in the art will understand that, due to the sustained-release properties of hydrogels and the differences in drug partition coefficients between the gel phase, liquid phase, and cellular phase, it is reasonable and expected that the nominal concentration of the encapsulated drug will be higher than the optimal concentration of the free drug. As shown in Examples 1, 2, and 6, the optimal treatment concentration of HG@OB in the encapsulated group was higher than that in the free group, but this difference did not lead to increased toxicity.

[0073] Example 8

[0074] Sterilized solutions of different concentrations (0, 40, 100 μM) of crotonyl glycoside B and polypeptide (final concentration 0.03 g / mL) were added to 24-well plates (100 μL per well). The plates were incubated at 37°C to allow the hydrogel (HG@OB) to form in situ within the wells. After solidification, bone marrow mesenchymal stem cells were seeded into the 24-well plates at a density of 2 x 10⁻⁶ m² / well. 4 After culturing for 48 hours, remove the plate, aspirate the culture medium, wash once with PBS, add 300 μL of Calcein / PI staining working solution (1.5 mL detection buffer + 1.5 μL Calcein-AM + 1.5 μL PI) to each well, incubate in the dark for 30 min, gently wash twice with 1 mL of PBS to each well, and observe the imaging effect under a fluorescence microscope (live cells will be stained green, and dead cells will be stained red).

[0075] like Figure 8 As shown, the Live / Dead experiment uses fluorescence excitation to cause live cells to emit green fluorescence and dead cells to emit red fluorescence, thus verifying the effect of oroxylum indicum B on cell activity and morphology. In the blank control group (CON), a large number of uniformly distributed green fluorescent signals were visible in the field of view, with only a very small number of red fluorescent spots, indicating that bone marrow mesenchymal stem cells maintained high activity, exhibited a typical spindle-shaped morphology, and were well-spread. In the 40μM HG@OB treatment group, the density of green fluorescent signals in the field of view was comparable to that of the control group, while the number of red fluorescent spots increased slightly but remained at a very low level, suggesting that this concentration of HG@OB has excellent cell compatibility, did not significantly inhibit the activity of bone marrow mesenchymal stem cells, and did not cause any abnormal changes in cell morphology. In the 100μM HG@OB treatment group, the density of green fluorescent signals in the field of view was significantly reduced, the number of red fluorescent spots was significantly increased, and the cell spreading area was reduced and the morphology was shrunken, indicating that high concentrations of HG@OB have significant cytotoxicity to bone marrow mesenchymal stem cells, significantly reducing cell activity and altering cell morphology. Live / Dead fluorescence staining results further validated the concentration-dependent regulatory effect of HG@OB on bone marrow mesenchymal stem cells (BMSCs) at the morphology and activity levels: low concentration (40 μM) HG@OB exhibited good cell compatibility and maintained high activity and normal morphology of BMSCs; high concentration (100 μM) HG@OB, however, showed significant cytotoxicity. These results are consistent with the aforementioned CCK-8 proliferation assays, providing direct morphological evidence for the safe application of HG@OB in bone tissue repair.

[0076] Example 9

[0077] The bone marrow mesenchymal stem cells were adjusted to a cell density of 5 × 10⁻⁶. 4Cells / mL, 24-well Transwell plates (8 μm pores) were used. Transwell chambers were placed in the wells, and 200 μL of the above bone marrow mesenchymal stem cell suspension was added to the upper chamber of each well. Control group (CON): 500 μL of basal culture medium (α-MEM) was added to the lower chamber; HG group: 500 μL of α-MEM culture medium containing blank hydrogel was added to the lower chamber; HG@OB group: 500 μL of α-MEM culture medium containing 100 μL of HG@OB hydrogel loaded with 40 μM oxycarpus bisacodyl B was added to the lower chamber. The Transwell plates were incubated at 37°C in a 5% CO2 incubator for 24 h to allow bone marrow mesenchymal stem cells to migrate towards the lower chamber. After incubation, remove the Transwell chambers, discard the liquid in the upper chamber, and gently wash twice with PBS. Immerse the chambers in 4% paraformaldehyde solution and fix at room temperature for 20 min. After washing twice with PBS, add 0.1% crystal violet staining solution to the upper chamber and stain at room temperature for 15 min. Slowly rinse the chambers with PBS, remove any unmigrated residual cells from the upper chamber with cotton swabs, blot the surface moisture with filter paper, and air dry upside down. Observe the stained Transwell chambers under an upright microscope.

[0078] The results are as follows Figure 9 As shown, in the control group (CON), the number of bone marrow mesenchymal stem cells migrating from the upper chamber to the lower chamber was relatively small, with only a few stained cells visible in the field of view; in the HG group, the number of migrating bone marrow mesenchymal stem cells was significantly increased, and the cells were densely distributed; in the HG@OB group, the number of bone marrow mesenchymal stem cells migrating from the lower chamber was similar to that in the HG group, indicating that HG has a significant recruitment (migration chemotaxis) effect on bone marrow mesenchymal stem cells, and HG@OB also has a recruitment effect.

[0079] Example 10

[0080] To evaluate the effects of different treatments on the osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs), this experiment included three treatment groups: a blank hydrogel control group (HG), a free oroxylin and oxyphylla glycoside B treatment group (OB, 5 μM), and an oroxylin and oxyphylla glycoside B-loaded hydrogel treatment group (HG@OB, loading concentration 20 μM). The specific steps are as follows:

[0081] BMSCs in 1x10 5 Cells were seeded in 12-well plates and cultured routinely at 37°C and 5% CO2. After cell attachment, the medium was replaced with osteogenic induction medium (containing 50 μg / mL vitamin C, 10 mM β-glycerophosphate, and 100 nM dexamethasone), and the cells were treated as follows:

[0082] HG group: Add blank self-assembled peptide hydrogel (100 μL);

[0083] OB group: Add free oroxylon ammodendron B to a final concentration of 5 μM;

[0084] HG@OB group: Add 100 μL of self-assembled polypeptide hydrogel loaded with 40 μM oroxylin B.

[0085] During the culture period, half of the cell culture medium was replaced every 3 days to maintain the induction environment and replenish the active ingredients.

[0086] (1) Five days after induction, the effect of early osteogenic differentiation was assessed by the BCIP / NBT alkaline phosphatase chromogenic method. The osteogenic induction medium in the cell culture plate was aspirated, the cells were washed twice with PBS, and an appropriate amount of 4% paraformaldehyde fixative was added to fix the cells at room temperature for 15–20 min. After aspirating the fixative, the cells were washed twice with PBS. According to the kit instructions, BCIP / NBT chromogenic solution A and solution B were mixed at a volume ratio of 100:1. After thorough mixing, an appropriate amount of chromogenic working solution was added to the culture wells. The culture plate was then incubated at room temperature in the dark for 15–30 min, during which the staining depth was observed under a microscope. When the purple precipitate reached the expected depth, the chromogenic working solution was immediately aspirated, and the cells were washed twice with deionized water to terminate the chromogenic reaction. The cell staining was observed under an inverted microscope, and typical fields of view were selected for imaging. The alkaline phosphatase (ALP) activity and the level of early osteogenic differentiation were assessed by the depth and distribution of the purple precipitate.

[0087] Alkaline phosphatase is an early marker enzyme for osteogenic differentiation, and its expression level can be visually reflected by the depth of staining precipitation; the deeper the staining, the higher the alkaline phosphatase activity and the stronger the osteogenic differentiation capacity. Results are as follows: Figure 10 As shown, the HG control group showed only a light purple staining in the field of view, without obvious dark precipitate, indicating that the blank hydrogel had no significant promoting effect on the early osteogenic differentiation of bone marrow mesenchymal stem cells. The staining depth of the free oroxylin B treatment group was significantly deeper than that of the HG control group, with uniformly distributed medium-intensity purple precipitate, suggesting that free oroxylin B can promote the early osteogenic differentiation of bone marrow mesenchymal stem cells. The HG@OB treatment group showed a denser, darker purple precipitate with a wider distribution, indicating that HG@OB can enhance the alkaline phosphatase activity of bone marrow mesenchymal stem cells, and its effect in promoting early osteogenic differentiation is superior to that of the blank hydrogel and free oroxylin B.

[0088] (2) After 14 days of induction, the osteogenic mineralization effect was assessed by Alizarin Red S staining. Osteogenic induction medium was aspirated from the cell culture plate, and the cells were washed twice with PBS. An appropriate amount of 4% paraformaldehyde fixative was added, and the cells were fixed at room temperature for 20 minutes. After aspirating the fixative, the cells were washed twice more with PBS. Alizarin Red S staining solution was added to the culture wells, ensuring complete coverage of the cell surface, and incubated at room temperature for 30 minutes. The staining solution was aspirated, and the cells were slowly washed three times with deionized water until no obvious red residue remained in the elution solution. At this point, the mineralized nodules would appear clearly red. The cell staining was observed under an inverted microscope, and typical fields of view were selected for imaging. The intensity of the red precipitate was used to assess the cell's mineralization capacity and late-stage osteogenic differentiation level.

[0089] Alizarin Red S can bind to calcium salts in the extracellular matrix to form a red precipitate. The staining depth, number, and area of ​​mineralized nodules directly reflect the mineralization capacity of cells and are a hallmark indicator of late-stage osteogenic differentiation. Results are as follows: Figure 11 As shown, in the HG control group, only scattered, light red precipitates were visible in the field of view, with few and small mineralized nodules. The staining depth of the free oroxylin B treatment group was significantly deeper than that of the HG control group, with more uniformly distributed red mineralized nodules visible, suggesting that free oroxylin B can promote the formation of mineralized nodules in bone marrow mesenchymal stem cells. The HG@OB treatment group showed dense, deep red precipitates in the field of view, with the largest number, largest area, and widest distribution of mineralized nodules, indicating that HG@OB can significantly promote late osteogenic differentiation and mineralization of bone marrow mesenchymal stem cells, and its effect is significantly better than that of blank hydrogel and free oroxylin B.

[0090] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. An orobanchyl B-loaded self-assembling polypeptide hydrogel, characterized in that, The hydrogel contains oroxylin B and a self-assembled polypeptide, the amino acid sequence of which is shown in SEQ ID No. 1, and the concentration of which is 0.02-0.05 g / mL; the concentration of oroxylin B is 5-40 μM, and the oroxylin B is in situ encapsulated in the three-dimensional nanofiber network structure formed by the self-assembled polypeptide.

2. The hydrogel according to claim 1, characterized in that, The hydrogel can mimic the bone tissue microenvironment in vivo, promoting the recruitment and proliferation of bone marrow mesenchymal stem cells.

3. The hydrogel according to claim 1, characterized in that, The hydrogel can upregulate the expression of osteogenic-related genes in bone marrow mesenchymal stem cells, inducing them to differentiate into osteoblasts.

4. A method for preparing a self-assembled polypeptide hydrogel loaded with oroxylin B as described in any one of claims 1-3, characterized in that, Includes the following steps: (1) Disperse oroxylum indicum B in phosphate buffer and dissolve by sonication in the dark to obtain working solution of oroxylum indicum B, wherein the concentration of oroxylum indicum B is 5-40 μM; (2) Add the self-assembled peptide to the working solution, shake to mix, and obtain a mixture; (3) The mixture is incubated at 30-40°C in the dark with shaking for 8-24 hours to obtain the hydrogel.

5. The preparation method according to claim 4, characterized in that, The pH of the phosphate buffer in step (1) is 7.2-7.4; the final concentration of the self-assembled polypeptide in step (2) is 0.02-0.05 g / mL.

6. The use of the self-assembled polypeptide hydrogel loaded with oroxylin B according to any one of claims 1-3 in the preparation of biomaterials for repairing bone defects.

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