Polypeptide dynamic co-assembled extracellular matrix bionic scaffold for directional differentiation of stem cells as well as preparation method and application of polypeptide dynamic co-assembled extracellular matrix bionic scaffold

Abstract

The invention provides a polypeptide dynamic co-assembled extracellular matrix bionic scaffold for directional differentiation of stem cells as well as a preparation method and application thereof, and belongs to the technical field of adaptive scaffold materials, the extracellular matrix bionic scaffold is formed by co-assembling polypeptide P1 and polypeptide P2 in water, the molar ratio of the polypeptide P1 to the polypeptide P2 is 1: 1, and the polypeptide P1 and the polypeptide P2 are co-assembled to form the extracellular matrix bionic scaffold. The amino acid sequence of the polypeptide P1 is as shown in SEQ ID NO.1, and the amino acid sequence of the polypeptide P2 is as shown in SEQ ID NO.2. The extracellular matrix bionic scaffold can dynamically adapt to cell behaviors, can more effectively regulate differentiation of stem cells, promote cell proliferation and guide the stem cells to be differentiated into bone formation, so that the quality of regenerated bones and the bone defect repair effect are improved.

Description

Technical Field

[0001] The invention belongs to the technical field of adaptive scaffold materials, and particularly relates to a polypeptide dynamically co-assembled extracellular matrix bionic scaffold for directed differentiation of stem cells, and a preparation method and application thereof. Background Art

[0002] Bone defects or functional impairments caused by traffic accidents, diseases, genetics, and aging remain major issues that affect the quality of human life and even individual survival. Recent advances in stem cell therapy and tissue engineering have shown great potential to advance medical treatment and improve patient outcomes. The extracellular matrix (ECM) is a key microenvironment for cell and tissue growth and has become a focus of tissue engineering research because of its role in mediating cell function. In addition to providing structural support, ECM also promotes signal transduction by interacting with cell surface receptors, and has unique advantages in bone repair such as promoting cell adhesion, proliferation, migration, and differentiation. Therefore, the application of ECM in tissue engineering has received increasing attention.

[0003] Over the past two decades, remarkable progress has been made in utilizing synthetic polymers as biomimetic scaffolds that mimic the properties of natural ECM. Specifically, the systematic optimization of scaffold properties, including stiffness and degradability, has attracted particular attention. These advances can create a static microenvironment that mimics natural ECM conditions, thereby enhancing cell attachment, proliferation, and function. However, stem cell differentiation involves a complex interplay of multiple cellular processes that require constant modification of the ECM to accommodate these dynamic changes. Recent advances have engineered mechanodynamic polymers that feature tunable cross-linking densities in response to a variety of stimuli, including light, chemical cues, and sound waves. Nonetheless, ensuring seamless cellular interactions with ECM-mimicking matrices during various cellular processes remains a pressing challenge that requires further investigation.

[0004] The existing patent technology "Hydrogel inclusion complex containing physiologically active substances bound to thermosensitive poly (phosphazene) by subject-guest interaction using β-cyclodextrin and its use" introduces a hydrogel inclusion complex based on thermosensitive poly (phosphazene) and β-cyclodextrin, which binds physiologically active substances through subject-guest interaction and has multiple potential application values, especially in the field of tissue regeneration and stem cell differentiation regulation. Its innovative subject-guest interaction mechanism and adjustable physiologically active substance loading capacity give it broad application prospects in the biomedical field. Hydrogels are widely used in tissue engineering due to their high biocompatibility and similarity to living tissues. However, traditional biocoupling technology has problems such as toxicity, low reproducibility and complex synthesis process. The researchers proposed a new type of hydrogel inclusion complex, which uses the subject-guest interaction of β-cyclodextrin to solve the above problems and achieve precise regulation of stem cell differentiation. The core of the invention is a hydrogel inclusion complex composed of thermosensitive poly (phosphazene), hydrophobic amino acids, hydrophilic polymers, β-cyclodextrin and polypeptides. Physiologically active substances act as guest molecules, bind to β-cyclodextrin through host-guest interactions, and can be slowly released. The complex is thermosensitive and transforms into a gel state at body temperature, providing favorable conditions for stem cell differentiation.

[0005] Although the hydrogel inclusion complex of the invention has shown significant advantages and innovations in tissue regeneration and stem cell differentiation regulation, there may still be some potential disadvantages or limitations in practical applications, such as poor stability of simple polypeptides in vivo, no regular structure, short half-life, and low efficiency. Although the differentiation of stem cells into specific cell types can be regulated by controlling the type and proportion of guest molecules, the accuracy of this regulation still needs to be further improved. In a complex in vivo environment, the differentiation of stem cells is affected by a combination of factors, including extracellular matrix, cell-to-cell interactions, growth factors, etc. Therefore, it may be difficult to achieve completely accurate stem cell differentiation regulation relying solely on the physiologically active substances in the hydrogel, and there may be problems such as low efficiency in the interaction between cells. The extracellular matrix is ​​an important signal that directly connects cells to cells. The ligands on the extracellular matrix can directly bind to the receptors on the cells, activate the relevant effectors in the cells, and transmit the extracellular signals to the nucleus through structures such as structural proteins to complete the regulation of genes. The following are the main functions of the extracellular matrix: the extracellular matrix provides physical support for cells, maintains the morphology of cells and the structural integrity of tissues. For example, the fiber network formed by collagen provides a tough support framework for cells, allowing cells to exist stably in specific locations and construct tissues and organs with specific morphology and functions. Regulating cell metabolism: The extracellular matrix can bind and store a variety of bioactive molecules such as growth factors and cytokines, and release these molecules at the appropriate time to regulate cell metabolic activities. For example, insulin-like growth factor binding protein can bind to insulin-like growth factor and store it in the extracellular matrix. When cells need it, it is released to promote cell growth and proliferation. Regulating cell proliferation and differentiation: The extracellular matrix interacts with receptors on the cell surface, activates intracellular signaling pathways, affects the expression and activity of cell cycle proteins, and thus regulates cell proliferation. During embryonic development, a specific extracellular matrix microenvironment induces stem cells to differentiate into different cell types. For example, neural stem cells differentiate into neurons, astrocytes, etc. in a specific extracellular matrix environment. Influencing cell migration: The composition and structure of the extracellular matrix determine the path and speed of cell migration. During tumor metastasis, tumor cells degrade the surrounding extracellular matrix to form channels that are conducive to their migration, and migrate to surrounding tissues or distant organs along the direction of the extracellular matrix fibers. Summary of the invention

[0006] In order to solve the above technical problems, the present invention provides a polypeptide dynamically co-assembled extracellular matrix bionic scaffold for directed differentiation of stem cells, which can dynamically adapt to cell behavior, more effectively regulate the differentiation of stem cells, promote cell proliferation, and guide stem cell differentiation towards osteogenesis, thereby improving the quality of regenerated bone and the effect of bone defect repair.

[0007] The present invention also provides a preparation method and application of a polypeptide dynamically co-assembled extracellular matrix bionic scaffold for directed differentiation of stem cells.

[0008] The present invention is achieved through the following technical solutions:

[0009] The present invention provides a polypeptide dynamically co-assembled extracellular matrix biomimetic scaffold for directed differentiation of stem cells. The extracellular matrix biomimetic scaffold is co-assembled by polypeptide P1 and polypeptide P2 in water, the molar ratio of the polypeptide P1 to the polypeptide P2 is 1:1, the amino acid sequence of the polypeptide P1 is shown in SEQ ID NO.1, and the amino acid sequence of the polypeptide P2 is shown in SEQ ID NO.2.

[0010] Based on the same inventive concept, the present invention provides a method for preparing a polypeptide dynamically co-assembled extracellular matrix biomimetic scaffold for directed differentiation of stem cells, the preparation method comprising:

[0011] Dissolving polypeptide P1 and polypeptide P2 in water, and performing co-assembly to obtain a reaction solution;

[0012] freeze-drying the reaction solution to obtain an extracellular matrix biomimetic scaffold;

[0013] The amino acid sequence of the polypeptide P1 is shown in SEQ ID NO.1, and the amino acid sequence of the polypeptide P2 is shown in SEQ ID NO.2.

[0014] Further, the molar ratio of the polypeptide P1 to the polypeptide P2 is 1:1;

[0015] In the reaction solution, the molar concentration of the polypeptide P1 is 200 μM.

[0016] Based on the same inventive concept, the present invention provides a polypeptide reagent for directed differentiation of stem cells, the polypeptide reagent comprising polypeptide P1 and polypeptide P2, the amino acid sequence of polypeptide P1 is shown in SEQ ID NO.1, and the amino acid sequence of polypeptide P2 is shown in SEQ ID NO.2.

[0017] Furthermore, the molar ratio of the polypeptide P1 to the polypeptide P2 is 1:1.

[0018] Based on the same inventive concept, the present invention provides the use of a polypeptide dynamically co-assembled extracellular matrix biomimetic scaffold for directed differentiation of stem cells in the preparation of an agent or drug for guiding the directed differentiation of stem cells.

[0019] Furthermore, the stem cells include bone marrow mesenchymal stem cells.

[0020] Based on the same inventive concept, the present invention provides the use of a polypeptide dynamically co-assembled extracellular matrix bionic scaffold for directed differentiation of stem cells in the preparation of a drug for repairing bone defects or bone function damage.

[0021] Based on the same inventive concept, the present invention provides the use of a polypeptide dynamically co-assembled extracellular matrix bionic scaffold for directed differentiation of stem cells in the preparation of bone regeneration drugs.

[0022] Based on the same inventive concept, the present invention also provides a drug for repairing bone defects or bone function damage, wherein the active ingredients of the drug include the above-mentioned polypeptide dynamically co-assembled extracellular matrix bionic scaffold for directed differentiation of stem cells;

[0023] Alternatively, the active ingredient of the drug includes the above-mentioned polypeptide agent for directed differentiation of stem cells.

[0024] One or more technical solutions in the embodiments of the present invention have at least the following technical effects or advantages:

[0025] 1. The polypeptide dynamic co-assembly extracellular matrix biomimetic scaffold for directed differentiation of stem cells of the present invention utilizes the characteristics of self-assembly and co-assembly and adopts in situ molecular assembly to make a tailored dynamic response to the ever-changing cellular microenvironment, which is essential for guiding stem cell differentiation and continuously replicating the complex cues inherent in natural tissue development. The extracellular matrix biomimetic scaffold exhibits remarkable adaptability through interaction with stem cell membranes and cells, and exhibits effective osteogenic differentiation. This innovation not only emphasizes the potential of bionic materials in regenerative medicine, but also emphasizes the importance of a flexible and interactive matrix in promoting therapeutic efficacy. The polypeptide scaffold can dynamically adapt to the changing needs of the cellular environment, which is a key step in the development of advanced biomaterials for clinical applications.

[0026] 2. The polypeptide dynamic co-assembly extracellular matrix bionic scaffold for directed differentiation of stem cells of the present invention has obvious advantages over traditional methods in the manufacture of simulated ECM materials. The ordered nanonetwork formed by the co-assembly of peptides P1 and P2 is very similar to the structure of natural ECM, providing a bionic microenvironment and effectively mimicking the natural tissue structure. In addition, the co-assembly process and conformational transition of peptides P1 and P2 replicate the dynamic characteristics of natural ECM and promote cell interactions that support optimal growth and function. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the following briefly introduces the drawings required for use in the description of the embodiments. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work.

[0028] Figure 1 Schematic representation of peptide coassembly to form nanobiointerfaces that modulate integrins to affect stem cell differentiation and bone regeneration.

[0029] Figure 2 Results of biophysical properties determination of P1 and P2: (a) TEM and SEM images of P1 (200 μM) self-assembled in water; (b) TEM and SEM images of P2 (200 μM) self-assembled in water; (c) TEM and SEM images of co-assembly of P1 and P2; (d) CD chromatograms of P1 (200 μM), P2 (200 μM), and a 1:1 mixture of P1 and P2; (e) HT graphs of P1 (200 μM), P2 (200 μM), and a 1:1 mixture of P1 and P2; (fi) SEM images of bone marrow mesenchymal stem cells after culturing P1, P2, and a 1:1 mixture of P1 / P2 in basal culture medium for 1 day.

[0030] Figure 3 Cell viability analysis diagram: (a) Live / dead staining of cells treated with polypeptides P1, P2 and a 1:1 mixture of P1 / P2 in basal medium for 1, 2 and 3 days; (b) WST-1 detection results of bone marrow mesenchymal stem cells after culturing with different concentrations of P1, P2 and P1 / P2 for 3 days.

[0031] Figure 4 Co-assembly of peptides P1 and P2 on the apical membrane of bone marrow mesenchymal stem cells: (a) light microscopy images treated with peptides P1, P2, and a 1:1 mixture of P1 / P2; (b) scanning electron microscopy (SEM) images treated with peptides P1, P2, and a 1:1 mixture of P1 / P2; (c) co-staining results with Congo red, actin green, and DAPI after culture on a collagen-coated surface with 1x PBS (control) and treated with P1, P2, or a 1:1 mixture of P1 / P2 at 200 μM for 3 days; (d) cross-sectional images of bone marrow mesenchymal stem cells treated with a 1:1 mixture of P1 / P2 at 200 μM for 3 days. Captured along the XZ plane and co-stained with Congo red, actin green, and DAPI.

[0032] Figure 5Figure 3 Representative images of alkaline phosphatase (ALP) staining in BMSCs treated with PBS (control), P1 (200 μM), P2 (200 μM), or a 1:1 mixture for 7 days to promote osteogenesis and inhibit adipogenesis in BMSCs in vitro: (a) Representative images of alkaline phosphatase (ALP) staining in BMSCs treated with PBS (control), P1 (200 μM), P2 (200 μM), or a 1:1 mixture for 7 days; (b) Quantitative analysis results of ALP activity in BMSCs on day 7; (c) Quantitative analysis results of ALP activity in BMSCs treated with PBS (control), P1 (200 μM), P2 (200 μM), or a 1:1 mixture for 7 days Representative images of Alizarin Red S staining in cells; (d) Quantitative analysis of BMSC mineralization on day 21; (eh) Relative expression levels of osteogenic-related genes RUNX2 (e), ALP (f), OCN (g), and SPP1 (h) in BMSCs on day 21; (i) Representative images of Oil RedO staining in BMSCs treated with PBS (control), P1 (200 μM), P2 (200 μM), or a mixture of P1 / P2 1:1; (j) Quantitative analysis of oil droplet formation in BMSCs on day 14; (km) Relative expression levels of adipogenesis-related genes PPARG (k), FABP4 (l), and LPL (m) in BMSCs on day 14.

[0033] Figure 6 To comprehensively analyze the molecular docking and dynamic simulation results of the peptides: their effectiveness in inducing mechanical transduction processes in bone marrow mesenchymal stem cells was demonstrated. Under equilibrium conditions, (a) and (b) show the schematic diagrams of the mesh processing of the surface of P1 and P2 in aqueous solution, where the main chain is represented by a strip; (c) The binding affinity ranking of P2 and P1 for integrin receptors was calculated based on the binding free energy calculated by the MM / PBSA method; (d) The root mean square fluctuation (RMSF) values ​​of the residues and different residues; (e) The color-coded RMSF map of the corresponding P2 and P1; highlighting the specificity (f) Fluorescence images showing the localization of YAP in bone marrow mesenchymal stem cells treated with PBS (control), P1 (200μM), P2 (200μM) or P1 / P21:1 mixture; (g) The scatter plot depicts the ratio of YAP intensity in the nucleus to the cytoplasm for each treatment group; (h) Western Blot analysis of the expression of phosphorylated YAP (p-YAP) and total YAP in BMSCs after treatment with PBS (control), P1 (200 μM), P2 (200 μM), or a 1:1 mixture of P1 / P2 in osteogenic medium.

[0034] Figure 7Molecular mechanism of peptide-mediated osteogenesis enhancement: (a) Volcano plot showing differentially expressed mRNAs between the P1 / P2 group and the control group from RNA sequencing data; (b) Heat map showing differentially expressed genes (DEGs) based on cluster analysis between P1 / P2 and control; (c) KEGG pathway enrichment analysis of DEGs between the P1 / P2 group and the control group; (d) GO function enrichment analysis of DEGs between the P1 / P2 group and the control group; (e) Heat map of upregulated cytokines in the MAPK pathway between the P1 / P2 group and the control group; (f) Heat map of upregulated cytokines in the PI3K-Akt pathway between the P1 / P2 group and the control group.

[0035] Figure 8 ECM-mimicking scaffolds can promote bone regeneration: (a) Representative microscopic CT images of femoral defects after different treatments: control group (Ctrl), P1, P2 and P1 / P2, microscopic CT images were taken at 4 weeks and 8 weeks after implantation, respectively; quantitative analysis of bone mineral density (BMD) (b), bone tissue volume to total tissue volume (BV / TV) (c) and trabecular number (Tb.N) (d) of femoral defects after 4 and 8 weeks of implantation.

[0036] Fig. 9 Histological analysis of femoral defects: (a) Histological staining (H&E staining) of histopathological bone tissues of different groups; b) Masson's trichrome histochemical staining of histopathological bone tissues of different groups; (cd) Quantitative analysis of Masson's trichrome histochemical staining of newly formed bone after 4 and 8 weeks. DETAILED DESCRIPTION

[0037] The present invention will be described in detail below in conjunction with specific implementations and examples, and the advantages and various effects of the present invention will be more clearly presented. It should be understood by those skilled in the art that these specific implementations and examples are used to illustrate the present invention, rather than to limit the present invention.

[0038] Throughout the specification, unless otherwise specifically stated, the terms used herein should be understood as meanings commonly used in the art. Therefore, unless otherwise defined, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art to which the present invention belongs. In the event of a conflict, the present specification takes precedence.

[0039] Unless otherwise specified, various raw materials, reagents, instruments and equipment used in the present invention can be purchased from the market or prepared by existing methods.

[0040] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0041] The polypeptide dynamic co-assembled extracellular matrix biomimetic scaffold for directed differentiation of stem cells of the present invention, as well as its preparation method and application will be described in detail below in conjunction with examples and experimental data.

[0042] Example 1

[0043] This example provides a method for preparing a polypeptide dynamically co-assembled extracellular matrix biomimetic scaffold for directed differentiation of stem cells and characterizes the same.

[0044] 1. Preparation method

[0045] The polypeptides used in the present invention are P1 (Lys-Arg-Ser-Arg-Phe-Phe-Phe-Ile-Lys-Leu-Leu-Ile) and P2 (Lys-Arg-Ser-Arg-Phe-Phe-Phe-Arg-Gly-Asp). The polypeptides were prepared by the SPPS method, and the required amount of peptides was dissolved in Milli-Q water to achieve a concentration of 200 μM as a stock solution for subsequent applications. Then, as needed, an appropriate volume of the stock solution was diluted with Milli-Q water or culture medium to prepare a working solution.

[0046] 2. Characterization and performance testing of co-assembled peptides

[0047] 1. For transmission electron microscopy (TEM) observations, briefly, 5 μL of the peptide solution was carefully deposited on a glow-discharged copper grid (400 mesh) coated with a carbon film, the excess solution was wiped off, and the grid was subsequently rinsed three times to meticulously eliminate any excess nanowires. Subsequently, the sample grid was negatively stained with 1.0% (w / v) uranyl acetate and allowed to air dry completely. TEM images were then acquired under high vacuum conditions using a JEM-1230R electron microscope at JEOL, Japan. To prepare samples for scanning electron microscopy (SEM), the peptide solution was carefully dispensed into a 35 mm glass-bottom dish. The sample was then frozen at -80 °C for 4 h to preserve its structure before imaging. This was followed by an overnight freeze-drying process using a high-efficiency freeze dryer (ES-2020, Hitachi, Japan) to ensure the preservation of the structural integrity of the sample. Prior to SEM observation, a thin layer of platinum was applied to the freeze-dried samples using a sputter coater (E1030, Hitachi, Japan) to improve the conductivity. Subsequent examinations were performed using a high-resolution SEM (JSM-7900F, JEOL, Japan) operating at an accelerating voltage of 3 kV. To prepare peptide-treated BMSCs for SEM observation, cells were first treated with the peptide assembly, and then the culture medium was carefully aspirated and the cells were washed three times with 1x PBS buffer. The cells were then fixed with 2.5% glutaraldehyde in 0.1 M calcium carbonate buffer for 30 min and then incubated with 1% osmium tetroxide (OsO) in the same buffer. 4 ) was fixed for 30 minutes. The cells were thoroughly washed with Milli-Q water for 5 minutes, and this process was repeated three times, and then gradually dehydrated through a series of graded ethanol solutions. Before scanning electron microscopy, all prepared samples were carefully coated with a 5nm thick osmium layer by an osmium coater (OPC80T, Filgen). Subsequently, high-resolution SEM images were taken at an accelerating voltage of 1.0 kV using a field emission scanning electron microscope (FE-SEM) (JSM-IT800SHL (JEOL, Japan)).

[0048] 2. Circular Dichroism (CD) Spectroscopy

[0049] The secondary structures of peptide assemblies in aqueous solution were investigated using a JASCO J-820 spectrophotometer (Japan Analytical Industry Co., Ltd.) under nitrogen. A quartz cuvette with a path length of 1.0 mm was used as the sample container. Spectral measurements were performed in the range of 190 to 250 nm using a precise 1 mm path length quartz cuvette to ensure accurate data acquisition. Each sample was scanned three times to ensure data accuracy.

[0050] 3. Cell Culture

[0051] The present invention uses bone mesenchymal stromal cells (BMSCs) extracted from the femoral bone marrow of SpraggeDawley (SD) rats. BMSCs are maintained in low-glucose Dulbecco's modified Eagle's medium (DMEM), to which 10% fetal bovine serum (FBS), 100 μg / mL streptomycin and 100 U / mL penicillin are added. Culture is carried out at 37°C in a 5% CO2 atmosphere, and the growth medium is renewed every 48 hours.

[0052] 4. Cell Viability Assay

[0053] For cell viability analysis, cells were first seeded into 96-well flat-bottom tissue culture plates at a precise tissue culture density (0.5 × 104 cells / cm 2 , and incubated overnight to ensure cell adhesion). The next day, the medium was replaced with osteogenic medium (10mM sodium β-glycerophosphate, 50ug / mL ascorbic acid, 10-8M dexamethasone) enriched with various peptides (P1, P2, P1 / P2) to evaluate their effects. For comparative analysis, control cells were cultured in osteogenic medium supplemented with an equal amount of phosphate-buffered saline (PBS). After 72 hours of incubation, cell viability was assessed using the WST-1 detection kit according to the manufacturer's instructions.

[0054] For live / dead viability assessment, designated sections of the above samples were further processed using calcein (AM) and propidium iodide (PI) staining. After staining, cell viability was assessed using fluorescence microscopy on a Nikon Eclipse Ts2R microscope to visualize live (green) and dead (red) cells.

[0055] 5. Alkaline Phosphatase and Alizarin Red S Staining Analysis

[0056] The early osteogenic differentiation and late calcium deposition of BMSCs induced by peptide-rich compounds (PBS, 200 μM P1, 200 μM P2, P1 / P2 to 500 μM) were evaluated by alkaline phosphatase (ALP) activity and Alizarin Red S staining, respectively. After 7 days of culture, BMSCs were fixed with 4% paraformaldehyde solution, stained with ALP activity kit for 30 min, and samples were captured by microplate reader. After 21 days of culture, Alizarin Red S (ARS) staining was performed to evaluate calcium deposition. BMSCs were fixed and treated with 0.1% ARS solution for 15 min, and images were recorded with a light microscope (Olympus). To quantify calcium deposition, BMSCs were incubated with 10% cetylpyridinium chloride, and the supernatant was collected and the absorbance was measured by microplate reader. Three samples were used for analysis.

[0057] 6. Oil Red O Staining and Lipid Droplet Quantification Analysis

[0058] To study adipogenic differentiation, bone marrow mesenchymal stem cells suspended with different compounds were cultured in 24-well culture plates containing adipogenic medium. After a 14-day incubation period, the cells were washed twice with preheated PBS, subsequently fixed with a 4% paraformaldehyde solution, and then briefly treated with a 60% 2-propanol solution for 5 minutes. Then soaked in Oil Red O working solution for another 5 minutes and then washed thoroughly with PBS. The images of the samples were captured under an optical microscope magnification of 10×. For quantitative analysis, the stained samples were air-dried and soaked in a 2-propanol solution to extract the Oil Red O dye. Then, the absorbance of each well was recorded using a multimode plate recorder. Three samples were used for analysis (n=3).

[0059] 7. Real-time PCR Analysis

[0060] The relative expression levels of RUNX2, ALP, OCN, SPP1 and adipogenesis-related genes PPARG, FABP4, and LPL in BMSCs were detected by RT-qPCR. BMSCs were cultured in osteogenic / adipogenic medium enriched with polypeptide compounds (PBS, 200 μM P1, 200 μM P2, P1 / P2-500 μM), respectively. After 21 days of incubation in osteogenic medium, mRNA was extracted using RNAiso+ kit, and then cDNA was synthesized using reverse transcription kit (TaKaRa, Japan). TBGreen qPCR (Takara, Japan) was used for qRT-PCR, and GAPDH was used as the internal control gene to detect the expression levels of osteogenic-related genes (RUNX2, ALP, OCN, and SPP1). The threshold cycle (Ct) method was used to calculate the expression level of mRNA (R = 2-ΔΔCt). Similarly, after 14 days of culture in adipogenic medium, the expression levels of adipogenic-related genes (PPARG, FABP4, LPL) were analyzed by qRT-PCR.

[0061] 8. Molecular Docking

[0062] The protein structure of integrin heterodimer was obtained from the Protein Data Bank. P1 and P2 were molecularly docked with integrins αVβ1, αVβ3 and αVβ6 and HADDOCK2. The resulting docking poses were analyzed using PyMol.

[0063] 9. Molecular dynamics simulation

[0064] Molecular dynamics simulations of peptide-integrin binding were performed using Gromacs with the CHARMM36m force field. Integrin-peptide heterodimers were solvated in a three-wire box. MD simulations were then performed at 310 K and 1 bar pressure with a time step of 30 ns using NPT integration, where the v-rescale algorithm and the Parrinello-Rachman thermostat were used for temperature coupling and pressure coupling, respectively. Van der Wall interactions were cut off from 0.8 to 1.2 nm using a force switch, and long-range Coulomb interactions were calculated using the Ewald method. Binding free energies were calculated using the molecular mechanics Poisson-Boltzmann surface area (MM / PBSA) method.

[0065] 10. Confocal Imaging

[0066] Bone marrow mesenchymal stem cells were seeded at a density of 1×10 cells per culture dish on a 35 mm glass bottom culture dish and cultured in an atmosphere of 5% CO2 at 37°C to promote cell adhesion. Then, the culture medium was updated with osteogenic medium containing 200 μM of the polypeptide compound. After 72 hours of culture, the culture medium was discarded and the cells were gently washed with preheated PBS. Subsequently, the cells were exposed to a freshly prepared Congo red solution at a concentration of 0.1 mg / mL in the culture medium and further cultured under the same conditions. After incubation, the cells were washed three times with PBS and then fixed with 4% paraformaldehyde (PFA) for 30 minutes. Nuclei and F-actin were visible by staining with 4', 6-diamino-2-phenylethylene (DAPI) and phenylpropanoid-alexa488 (Invitrogen, CA) for 30 minutes.

[0067] Immunofluorescence staining was performed using a YAP-specific primary antibody. Co-stained with DAPI and phalloidin-Alexa 488, fluorescent images were captured using a laser scanning microscope (Nikon A1 and LSM780, Zeiss). ImageJ software was used to quantify the nuclear-cytoplasmic distribution ratio (Nuc / cell ratio) of YAP, and the calculation formula was as follows:

[0068] YAP = Inuc / Anuc (total - Inuc) / (total - Anuc); Inuc is the integrated density of the nuclear region, Anuc is the area of ​​the nuclear region; Itotal is the total integrated density of the cell; Atotal is the total area of ​​the cell.

[0069] 11. Western Blot Analysis

[0070] Protein samples were separated using SDS-PAGE on 12.5% ​​polyacrylamide gels and then transferred to PVDF membranes (Millipore, Bedford, MA). The cell membranes were then probed with primary antibodies overnight at 4°C. Subsequently, horseradish peroxidase-conjugated secondary antibodies (diluted 1:5000) were used for detection and reacted with specific primary antibodies for 1 hour. Antibody reactivity was detected using an enhanced chemiluminescence (ECL) Western blotting system (Amersham Biosciences). ImageJ software was used to quantitatively evaluate the grayscale values ​​associated with each band.

[0071] 12. RNA Sequence Analysis

[0072] Cells were cultured in 6-well plates and then treated with P1 / P2 or fibronectin for 10 days. Subsequently, cells were collected and total RNA was extracted using TRIzol reagent (Thermo Fisher Scientific, USA). RNA sequencing services were provided by Zaigen Gene Technology Co., Ltd. (Beijing). Differential gene expression analysis was performed using the DESeq2 software package. Genes were classified as differentially expressed if they met the criteria of an adjusted P value < 0.05 determined by DESeq2.

[0073] 13. Surgical Procedures in Animal Models

[0074] The osteogenic repair effect was evaluated using an in vivo rat model, in which a defect was created in the femoral ostium. All experimental procedures were performed in accordance with the guidelines of the Animal Experiment Center of Zhengzhou University (approval number: ZZU-LAC2022111) and ethical approval was obtained from the relevant committees. A total of 24 male Sprague-Dawley rats were randomly divided into the control, P1, P2, and P1 / P24 groups. To reduce intraoperative complications, the rats were fasted for 12 h and further fasted for 4 h before surgery. After anesthesia was achieved by isoflurane inhalation, the rats were placed in a prone position and analgesia was given subcutaneously with meloxicam (0.1 mg / kg). The surgical area was prepared by shaving the hair and disinfecting the exposed skin with iodine phenol. A 15 mm incision was made and the distal femur was exposed by subperiosteal dissection. A 3 mm diameter drill was used to create the femoral defect. BMSC spheroids were injected into the defect site with sterile solution containing scaffold-free BMSC spheroids, P1-treated BMSC spheroids, P2-treated BMSC spheroids, and P1 / P2-treated BMSC spheroids. The opening was sealed with bone wax to prevent leakage. The incision was sutured with 3-0 silk. After surgery, rats were given preventive treatment with penicillin sodium (4 IU / kg, intramuscular injection) and meloxicam (0.1 mg / kg, subcutaneous injection) for 3 days to promote recovery.

[0075] 14. Micro-CT and Histological Analysis

[0076] SD rats were killed 4 and 8 weeks after implantation, and the femurs were removed and fixed with 4% paraformaldehyde. Subsequently, three-dimensional images were captured using a micro-computed tomography (micro-CT) scanner and reconstructed using NRecon software. In addition, new bone formation parameters (BMD, BV / TV, TB.N) were analyzed using CTAn software.

[0077] The femurs were decalcified in 10% EDTA solution (Servicebio, China) for 4 weeks. After decalcification, the samples were dehydrated by gradient ethanol, embedded in paraffin and sectioned. H&E staining and Masson's trichrome staining were used to evaluate the morphology of cells and tissues. Images were quantitatively analyzed using Image J software.

[0078] 3. Characterization and Performance Test Results

[0079] 1. Design and Characterization of Co-Assembling Peptides

[0080] In order to prepare P1 (Lys-Arg-Ser-Arg-Phe-Phe-Phe-Ile-Lys-Leu-Leu-Ile) and P2 (Lys-Arg-Ser-Arg-Phe-Phe-Phe-Arg-Gly-Asp) so that they can spontaneously assemble under relatively low concentration water conditions, we introduced the hydrophobic unit phenylalanine into the N-terminus of these peptides. This modification is intended to promote self-assembly driven by π-π interactions and hydrogen bonds. According to this strategy, P1 was obtained for self-assembly in aqueous solution). Following the same design principle, P2 (peptide 2, P2) was synthesized. Their self-assembly properties were further explored, and the results are shown in Figure 2 a and b. Unlike P2, P1 self-assembled into a soft hydrogel structure at a concentration of 200 μM in 1× PBS buffer, whereas P2 remained in a sol state at this concentration. Therefore, P1 showed a significant increase in hydrophilicity and underwent spontaneous self-assembly compared to P2. Transmission electron microscopy (TEM) and scanning electron microscopy (SEM) imaging further confirmed these findings, revealing that P1 self-assembled into elongated fibers that were arranged into thicker, stronger bundles. In contrast, P2 formed shorter, less organized fibers, indicating a lesser tendency to form complex fiber bundles.

[0081] Using electron microscopy imaging ( Figure 2 c) and circular dichroism (CD) analysis ( Figure 2d and e) The co-assembly of peptides P1 and P2 was investigated. TEM and SEM images showed significant morphological changes, with the self-assembly of P1 forming wider, straighter fibers, while the co-assembly of P1 and P2 produced thinner, more curved fibers. The CD spectrum of the P1 / P2 co-assembly exhibited a distinct signal that was clearly different from that of the individual self-assemblies of P1 and P2. This difference could be due to a change in the position of the assembled building blocks, providing further evidence for the co-assembly between P1 and P2.

[0082] 2. Co-assembly of peptides regulates cellular functions

[0083] To further investigate the effects of adaptive ECM mimics on the behavior of MSCs, we treated cells with peptides P1, P2, or a co-assembled P1 / P2 mixture at a concentration of 200 μM, significantly below the gelation concentration. This treatment led to the formation of extracellular scaffolds that adhered to the apical membrane of MSCs or were distributed between cells.

[0084] Our results showed that after 3 days of culture, cells treated with P1 or the coassembled P1 / P2 mixture exhibited varying degrees of aggregation, whereas cells treated with P2 alone showed minimal changes ( Figure 4 a). Further analysis using scanning electron microscopy and confocal fluorescence microscopy revealed that the assembled polypeptides not only formed on the surface of BMSCs, but also formed supramolecular fibers in the intercellular space ( Figure 4 bd). Remarkably, cells treated with the co-assembled P1 / P2 mixture underwent remodeling leading to the formation of multilayered cellular structures. This observation highlights the potential of these assemblies in directing cellular organization and function.

[0085] Alkaline phosphatase (ALP) is an early marker of osteogenic differentiation. We further studied the effects of biostimulatory peptides on bone marrow mesenchymal stem cell osteogenesis and the results showed that the co-assembly of P1 and P2 triggered a significant enhancement of ALP activity ( Figure 5 a and b). In addition, the P1 / P2 co-assembled scaffolds effectively promoted calcium deposition in the Alizarin Red S staining assay ( Figure 5 c and d), which is a key marker of late osteogenic differentiation. We also examined the expression levels of genes associated with osteogenic differentiation ( Figure 5 e). Similarly, P1 / P2 co-assembled scaffolds significantly upregulated bone markers, including runt-related transcription factor 2 (RUNX2) ( Figure 5 e), alkaline phosphatase (ALP) Figure 5 f), osteocalcin (OCN) Figure 5 g) and secretory phosphoprotein 1 (SPP1) ( Figure 5h), indicating the promotion of osteogenesis through the co-assembly contribution of P1 and P2.

[0086] 3. The co-assembly of peptides induced mechanical transduction of bone marrow mesenchymal stem cells.

[0087] To better understand the relationship between extracellular scaffold formation and phenotypic responses after BMSC treatment, we used molecular docking and molecular dynamics (MD) simulations to investigate the interactions between different ligands and integrin receptors. Figure 6 a and Figure 6 b shows a ribbon representation of the main-chain conformations of P1 and P2, illustrating the structural features of the peptides in a biologically relevant solvent environment.

[0088] Compared with P1, P2 has a significantly stronger binding affinity to integrin β and integrin αvβ6 receptors. In contrast, P1 is able to bind to a wider range of integrin receptors ( Figure 6 c) These unique features of the two peptides may underpin the enhanced cellular interactions observed following co-assembly treatment of P1 and P2, potentially amplifying their synergistic effects on cellular responses.

[0089] The root mean square fluctuation (RMSF) analysis method based on molecular dynamics simulation was used to evaluate the molecular stability of P1 and P2, which can show the structural stability and flexibility of each amino acid residue in the sequence. The RMSF value of each amino acid in the P1 and P2 molecules is shown in Figure 2. Figure 6 d and 6e. The red-highlighted P2 peptide chain has the highest RMSF value of about 3A, indicating significant structural fluctuations during the simulation. In contrast, the P2 peptide exhibits a more stable conformation and minimal structural motion, indicating greater chain rigidity. This enhanced rigidity may promote ordered self-assembly, which is consistent with our TEM observations.

[0090] Nuclear translocation of YAP is a key indicator of cellular mechanotransduction, particularly with regard to the crosstalk between external cues and integrin receptors. Consistent with reported studies, our results demonstrated that P1 and P2 co-assembly treatment resulted in significant changes in the cellular dynamics of YAP, leading to enhanced translocation to the nucleus ( Figure 6 f and g). Western blot (WB) analysis further confirmed this result ( Figure 6 h). Compared with the P1 and P2 groups, the coassembly of P1 / P2 promoted the phosphorylation of YAP in cells, subsequently triggering its translocation into the nucleus. This nuclear localization of YAP has a stronger promoting effect on osteogenesis. In summary, the coassembly of P1 / P2 on the apical membrane modulates the behavior of BMSCs and induces higher contractile forces than fibronectin coating and single assembly peptides P1 and P2.

[0091] 4. In vivo bone regeneration via ECM mimics

[0092] To further demonstrate the potential of ECM mimics in promoting bone repair in vivo, we chose a femoral defect model to evaluate bone regeneration performance. Bone marrow MSC spheroids pretreated with ECM mimics were implanted into the defect site. After the indicated time intervals, bone specimens were harvested for comprehensive radiological and histological analysis. All rats remained in good health and showed no signs of wound complications during the 4- and 8-week experimental periods. X-ray micro-computed tomography (micro-CT) images, such as Figure 8 a, depicts the changes in the defect site 4 and 8 weeks after implantation.

[0093] In the control group, BMSC spheroids were implanted without scaffolds, and a small amount of new bone formation was observed. In contrast, the P1 group, which received P1-treated BMSC spheroids, showed bone tissue regeneration at the defect edges after 4 weeks. Notably, after 8 weeks, the P1 group showed substantial progress, with a large amount of bone tissue filling the central cavity. The most obvious bone regeneration occurred in the P1 / P2 group, which involved the implantation of BMSC spheroids co-assembled with P1 and P2 into interpenetrating scaffolds. In this group, extensive bone regeneration was observed at the defect site after 4 weeks, and by 8 weeks, the defect was almost completely healed.

[0094] Newly regenerated bone at the defect site was quantified in detail, focusing on key parameters such as bone mineral density (BMD) ( Figure 8 b) Bone volume fraction (BV / TV) Figure 8 c) and trabecular number (Tb.N) ( Figure 8 d). Eight weeks after implantation, the BMD values ​​of the P1 and P1 / P2 groups were 116.5 mg / cm 3 and 139.7 mg / cm 3 , BV / TV percentages were 33.6% and 47.0%, and Tb.N measurements were 0.6mm-1 and 1.0mm-1, respectively. These values ​​were significantly higher than those in the control and P2 groups, with the P1 / P2 group showing the most significant improvements in all parameters.

[0095] Hematoxylin-eosin and Masson staining were further performed to detect the microstructure of the regenerated bone ( Fig. 9 a and b). Quantitative analysis of Masson staining is also shown in Fig. 9c and d. Consistent with our previous observations, the P1 / P2 group showed greater newly formed bone and tissue regeneration compared with the other groups. Four weeks after implantation, new mineralized bone structures covered the central area of ​​the defect cavity. After 8 weeks, the thickness of the new bone increased significantly. Abundant connective fibrous tissue, as well as scattered bone-like masses and osteocytes were seen in the P1 / P2 group. At the same time, primary organs (heart, liver, spleen, lung, and kidney) of patients in each group were collected for histological analysis. There were no significant changes in the other groups compared with the control group that did not receive peptide treatment. These results indicate that the scaffolds used in each group have good biosafety.

[0096] Finally, it should be noted that the terms "comprises," "includes," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that includes a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements that are inherent to such process, method, article, or apparatus.

[0097] Although the preferred embodiments of the present invention have been described, those skilled in the art may make other changes and modifications to these embodiments once they have learned the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention.

[0098] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.

Claims

1. A polypeptide dynamically co-assembled extracellular matrix biomimetic scaffold for directed differentiation of stem cells, characterized in that: The extracellular matrix biomimetic scaffold is co-assembled by polypeptide P1 and polypeptide P2 in water, the molar ratio of polypeptide P1 to polypeptide P2 is 1:1, the amino acid sequence of polypeptide P1 is shown in SEQ ID NO.1, and the amino acid sequence of polypeptide P2 is shown in SEQ ID NO.

2.

2. The method for preparing the polypeptide dynamically co-assembled extracellular matrix biomimetic scaffold for directed differentiation of stem cells according to claim 1, characterized in that: The preparation method comprises: Dissolving polypeptide P1 and polypeptide P2 in water, and performing co-assembly to obtain a reaction solution; freeze-drying the reaction solution to obtain an extracellular matrix biomimetic scaffold; The amino acid sequence of the polypeptide P1 is shown as SEQ ID NO.1, and the amino acid sequence of the polypeptide P2 is shown as SEQ ID NO.

2.

3. The method for preparing the polypeptide dynamically co-assembled extracellular matrix biomimetic scaffold for directed differentiation of stem cells according to claim 2, characterized in that: The molar ratio of the polypeptide P1 to the polypeptide P2 is 1:1; In the reaction solution, the molar concentration of the polypeptide P1 is 200 μM.

4. A polypeptide reagent for directed differentiation of stem cells, characterized in that: The polypeptide reagent includes polypeptide P1 and polypeptide P2. The amino acid sequence of polypeptide P1 is shown as SEQ ID NO.1, and the amino acid sequence of polypeptide P2 is shown as SEQ ID NO.

2.

5. The polypeptide reagent for directed differentiation of stem cells according to claim 3, characterized in that: The molar ratio of the polypeptide P1 to the polypeptide P2 is 1:

1.

6. Use of the polypeptide dynamically co-assembled extracellular matrix biomimetic scaffold for directed differentiation of stem cells as claimed in claim 1 in the preparation of reagents or drugs for guiding directed differentiation of stem cells.

7. The use according to claim 6, characterized in that: The stem cells include bone marrow mesenchymal stem cells.

8. Use of the polypeptide dynamically co-assembled extracellular matrix biomimetic scaffold for directed differentiation of stem cells as claimed in claim 1 in the preparation of drugs for repairing bone defects or bone function damage.

9. Use of the polypeptide dynamically co-assembled extracellular matrix biomimetic scaffold for directed differentiation of stem cells as claimed in claim 1 in the preparation of bone regeneration drugs.

10. A drug for repairing bone defects or bone function damage, characterized in that: The active ingredient of the drug includes the polypeptide dynamically co-assembled extracellular matrix biomimetic scaffold for directed differentiation of stem cells according to claim 1; Alternatively, the active ingredient of the drug includes the polypeptide agent for directed differentiation of stem cells according to claim 4 or 5.

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