Biological material for restoring ovarian functions, preparation method therefor and use thereof

Abstract

Provided are a biological material for restoring ovarian functions, a preparation method therefor and the use thereof, relating to the technical field of biological materials. A large number of experiments verify that: it is found that a specific biological protein component for reversing ovarian functions in extracellular vesicles is the growth differentiation factor 9 (GDF-9), and the mechanism of action thereof for restoring the ovary is determined, and therefore extracellular vesicles (GOEs) that overexpress GDF-9 serve as an active substance, and by means of extraction and purification of said component, the purpose of targeted therapy is achieved.

Description

Biomaterials, preparation methods and applications for repairing ovarian function Technical Field

[0001] This invention relates to the field of biomaterials technology, and in particular to a biomaterial for repairing ovarian function, its preparation method, and its application. Background Technology

[0002] Ovarian function plays a decisive role in female reproductive health. When ovarian failure occurs, a woman's fertility declines significantly. This is characterized by follicular atresia, leading to anovulation, infertility, and low sex hormone levels, severely impacting a woman's physical and mental health. Currently, the classic clinical treatment is hormone replacement therapy (HRT), which alleviates perimenopausal symptoms by exogenously supplementing estrogen and progesterone. However, HRT cannot improve follicular arrest or premature atresia, nor can it induce ovulation or improve female fertility; therefore, it cannot fundamentally repair the failing ovaries. Thus, how to repair ovarian function, restart follicle development, maturation, ovulation, sex hormone secretion, and improve fertility, is a key challenge and focus in the treatment of ovarian failure. Regenerative medicine offers new hope for treating ovarian failure. Mesenchymal stem cell therapy for ovarian failure has been validated, but the immune and tumor responses induced by stem cells limit its clinical application.

[0003] In view of this, the present invention is hereby proposed. Summary of the Invention

[0004] One of the objectives of this invention is to provide a biomaterial for repairing ovarian function, so as to at least solve one of the technical problems existing in the prior art.

[0005] The second objective of this invention is to provide a method for preparing the above-mentioned biomaterials for repairing ovarian function.

[0006] The third objective of this invention is to provide the use of the above-mentioned biomaterial for repairing ovarian function in the preparation of products for repairing ovarian function.

[0007] In order to achieve the above-mentioned objectives of the present invention, the following technical solution is adopted:

[0008] This invention provides a biomaterial for repairing ovarian function, comprising extracellular vesicles overexpressing GDF-9 as active substances.

[0009] Furthermore, the biomaterial is a membrane-forming agent;

[0010] Preferably, the biomaterial is in tablet, powder, emulsion, or liquid form.

[0011] This invention also provides a method for preparing the above-mentioned biomaterial for repairing ovarian function, comprising the following steps:

[0012] (a) Preparation of extracellular vesicles overexpressing GDF-9;

[0013] (b) The GDF-9 overexpressing extracellular vesicles obtained in step (a) are prepared as the biomaterial for repairing ovarian function.

[0014] Further, step (a) includes inducing GDF-9 overexpression in mesenchymal stem cells with GDF-9 mRNA, collecting cell supernatant, and obtaining GDF-9 overexpressing extracellular vesicles;

[0015] The sequence of the GDF-9 mRNA is Homo sapiens growth differentiation factor 9 (GDF9), RefSeqGene on chromosome 5, Sequence ID: NG_047051.1:5416-10700;

[0016] Preferably, the method for inducing GDF-9 overexpression in mesenchymal stem cells is selected from cell transfection or electroporation.

[0017] Further, step (b) includes injecting the GDF-9 overexpressing extracellular vesicles prepared in step (a) as the inner aqueous phase of 0.1% polyvinyl alcohol into dichloromethane containing PLGA as the oil phase, sonicating them, and then injecting them into a solution of 0.7% PVA. After emulsification, the biomaterial for repairing ovarian function is obtained.

[0018] Furthermore, prior to step (b), the method includes preparing the overexpressing GDF-9 extracellular vesicles into a lyophilized powder.

[0019] Furthermore, the present invention also provides the application of the above-mentioned biomaterial for repairing ovarian function, or the biomaterial for repairing ovarian function prepared by the above-mentioned preparation method, in the preparation of products for repairing ovaries.

[0020] Furthermore, the overexpression of GDF-9 extracellular vesicles in the biomaterials binds to the ACVR1B receptor in ovarian cells, causing SMAD2 to phosphorylate and enter the nucleus, thereby repairing the ovary.

[0021] Compared with the prior art, the present invention has the following beneficial effects:

[0022] This invention, through extensive experimental verification, has discovered that the specific biological protein component in extracellular vesicles that reverses ovarian function is Growth Differentiation Factor 9 (GDF-9), and its mechanism of action in repairing the ovary is based on this. Furthermore, using GDF-9-overexpressing extracellular vesicles as the active substance, this component is effectively extracted and purified to achieve targeted therapy. Simultaneously, this invention also found that when GDF-9-overexpressing extracellular vesicles are prepared as a membrane-based agent, they are more likely to rupture and release the effective molecules upon reaching tissues and organs. Attached Figure Description

[0023] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0024] Figure 1 is a schematic diagram illustrating the theoretical basis for the experimental examples of this invention.

[0025] Figure 2 is a schematic diagram of EVs derived from umbilical cord mesenchymal stem cells provided in the experimental example of the present invention; wherein, a, a schematic diagram of the isolation and purification of EVs; b, transmission electron microscopy showing the integrity and structure of EVs; c, Western blot detection of the expression of CD63 and CD9 in EVs.

[0026] Figure 3 shows the results of NTA detection of EV concentration and size provided in the experimental example of the present invention.

[0027] Figure 4 is a schematic diagram of the potential in vitro DO model for improving the survival ability of GCs by EVs, provided in the experimental example of the present invention.

[0028] Figure 5 shows the flow cytometry detection of GC activity, apoptosis, and death provided in the experimental example of this invention.

[0029] Figure 6 shows the flow cytometry quantitative effect of EVs in in vitro treatment of DO provided in the experimental example of this invention; n=3, data are expressed as mean±SD; * and ** indicate statistical significance between the two groups, ns indicate no statistical significance between the two groups. The viable cell rate in the EVs treatment group was significantly higher than that in the non-EVs treatment group, while the dead cell rate and apoptotic cell rate were significantly lower in the EVs treatment group. There was no significant difference in mechanical damage among the blank control group, the DO group, and the EVs treatment group.

[0030] Figure 7 shows the results of the effect of GDF-9 on the survival and proliferation of GCs in EV treatment provided by the experimental example of the present invention; where a, a schematic diagram of in vitro GC induction; b, the number of GCs increased after EV treatment; c, the cell count of GCs overexpressing GDF-9 was higher than that of the control group, and the cell count of GCs inhibited by GDF-9 was lower than that of the control group; df, TUNEL assay showed that apoptosis of GDF-9 GCs was reduced compared with EV-treated si-GDF-9 GCs; eg, compared with EV-treated si-GDF-9 GCs, the expression level of Ki-67 in GDF-9 GCs was higher.

[0031] Figure 8 shows the verification results of ACVR1B as a major regulator of GCs provided in the experimental examples of this invention; where: a) is a schematic diagram of the rat OD model, obtained through total RNA sequence sampling and analysis; b) KEGG pathway enrichment reveals the signaling pathway of cytokine-cytokine receptor interaction; c) Volcano plot of RNA-seq analysis; de) Western blot detection of GDF-9, ACVR1B, SMAD2 levels and SMAD2 phosphorylation status; fg) Western blot detection shows that inhibition of the GDF-9 gene leads to the inability of EVs to stimulate GDF-9 signal transduction, resulting in decreased expression levels of ACVR1B and SMAD2 / p-SMAD2 proteins.

[0032] Figure 9 shows the schematic diagram and results of the GOEs and animal experimental design provided in the experimental examples of this invention.

[0033] Figure 10 shows the verification results of how GDF-9 overexpressed EVs (GOEs) provided in the experimental example of this invention can improve ovarian function in DO rats. The rat body weight results are shown before modeling and after drug administration.

[0034] Figure 11 shows the verification results of GDF-9 overexpressing EVs (GOEs) improving ovarian function in DO rats provided in the experimental example of this invention; where ad represents the ovarian size and body weight of the rats after treatment; e represents the ovarian histology after treatment; the top row shows representative parts, with black boxes indicating enlarged areas; arrows represent PMF (primordial follicles), arrows represent SCF (secondary follicles), and asterisks represent ATF (atretic follicles); fh represents the number of follicles in the control group, DO, and GOEs treatment group; n=3, and data are expressed as mean ± SD.

[0035] Figure 12 shows the results of using follicle-stimulating hormone receptor (FSHR) as a biomarker for detecting ACVR1B-expressing GCs after GOE treatment, as provided in the experimental example of this invention; where a, IF detection of FSHR expression in ovarian GCs; b, IF detection of ACVR1B expression in ovarian GCs; c, IF detection of FHSR and ACVR1B expression in ovarian GCs; df, the number of FSHR and ACVR1B in the control group, DO and GO treatment groups and their expression levels at the mean fluorescence intensity; n=3, and data are expressed as mean ± SD.

[0036] Figure 13 shows the combined results of immunohistochemical (IHC) and immunofluorescence (IF) analyses provided in the experimental examples of this invention; where a) is the ovarian ACVR1B immunohistochemical image, with the black dashed box emphasizing the magnified area and the black arrow pointing to the positive result; b) is the ovarian ACVR1B immunofluorescence image, with the white dashed box emphasizing the magnified area and the white arrow pointing to the red fluorescent positive result; c) and d) are the quantitative analysis images of 13a and b, indicating that the expression of ACVR1B in the ovaries of DO rats treated with GOEs is higher than that in DO rats not treated with GOEs. Detailed Implementation

[0037] Unless otherwise defined herein, the scientific and technical terms used in conjunction with this invention shall have the meanings commonly understood by one of ordinary skill in the art. The meaning and scope of terms shall be clear; however, in any case of potential ambiguity, the definitions provided herein shall prevail over any dictionary or foreign definitions. In this application, unless otherwise stated, the use of "or" means "and / or". Furthermore, the use of the term "comprising" and other forms is non-limiting.

[0038] Generally, the nomenclature and techniques used in cell and tissue culture, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry and hybridization, together with those described herein, are those well-known and commonly used in the art. Unless otherwise stated, the methods and techniques of the present invention are generally carried out according to conventional methods well-known in the art and described in various general and more specific references, which are cited and discussed throughout this specification. Enzymatic reactions and purification techniques are carried out according to the manufacturer's instructions, as commonly practiced in the art, or as described herein. The nomenclature, laboratory procedures, and techniques used in analytical chemistry, synthetic organic chemistry, and medical and medicinal chemistry, together with those described herein, are those well-known and commonly used in the art.

[0039] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0040] According to one aspect of the present invention, a biomaterial for repairing ovarian function is provided, comprising a membrane formulation containing GDF-9 overexpressing extracellular vesicles as active substances.

[0041] Through extensive experimental verification, this invention has discovered that the specific biological protein component in extracellular vesicles that reverses ovarian function is growth differentiation factor 9 (GDF-9), which repairs the ovary. Furthermore, by using extracellular vesicles that overexpress GDF-9 as the active substance, this component is effectively extracted and purified to achieve the purpose of targeted therapy (Figure 1).

[0042] Since lipids are the membrane form of exosomes themselves, which is more in line with biological characteristics and can easily be compatible with cells in tissues and organs to achieve targeted therapy, when GDF-9 overexpressing extracellular vesicles are prepared as membrane-form agents, they are more likely to rupture membranes and release effective molecules after reaching tissues and organs. Therefore, in some preferred embodiments, the biomaterial is a membrane-form agent.

[0043] Optionally, the biomaterial is in tablet, powder, emulsion, or liquid form. For better clinical application, the biomaterial is preferably in tablet form. After extracellular vesicles form a film, they need to be cryopreserved to maintain stability. When placed in the pelvic cavity, the tablet form can directly adhere to the ovarian surface, and the extracellular vesicles become active upon contact with water and when the temperature rises to body temperature.

[0044] According to a second aspect of the present invention, a method for preparing the above-mentioned biomaterial for repairing ovarian function is provided, comprising the following steps:

[0045] (a) Preparation of extracellular vesicles overexpressing GDF-9;

[0046] (b) The GDF-9 overexpressing extracellular vesicles obtained in step (a) are prepared as the biomaterial for repairing ovarian function.

[0047] In some specific implementations, step (a) includes inducing GDF-9 overexpression in mesenchymal stem cells with GDF-9 mRNA, collecting cell supernatant, and obtaining GDF-9 overexpressing extracellular vesicles.

[0048] The sequence of the GDF-9 mRNA is Homo sapiens growth differentiation factor 9 (GDF9), RefSeqGene on chromosome 5, Sequence ID: NG_047051.1:5416-10700.

[0049] The method for inducing GDF-9 overexpression in mesenchymal stem cells is not limited; any conventional molecular biology method in the field that can achieve the induction function, such as cell transfection or electroporation, is acceptable. As a preferred option, electroporation can be used instead of stem cell transfection, which can effectively improve the efficiency of carrying effective molecules.

[0050] In some specific embodiments, step (b) includes injecting the GDF-9 overexpressing extracellular vesicles prepared in step (a) as the inner aqueous phase of 0.1% polyvinyl alcohol into dichloromethane containing PLGA as the oil phase, sonicating them, and then injecting them into a solution of 0.7% PVA. After emulsification, the biomaterial for repairing ovarian function is obtained.

[0051] Specifically, GDF-9 overexpressing extracellular vesicles were injected as the inner aqueous phase of 0.1% polyvinyl alcohol (PVA) into a PLGA-containing dichloromethane (DCM) oil phase. The entire mixture was then sonicated at 4°C. Immediately afterward, the emulsion was injected into a 0.7% PVA solution. The secondary emulsion was emulsified for 12 min on a high-speed homogenizer to obtain the final water / oil / water (w / o / w) mixture, which is the biomaterial for repairing ovarian function.

[0052] Optionally, before step (b), the method further includes preparing the GDF-9 overexpressing extracellular vesicles into a lyophilized powder. The lyophilized powder form offers advantages such as ease of carrying and transportation, enhanced stability, and convenient clinical use.

[0053] According to a third aspect of the present invention, the present invention also provides the application of the above-described biomaterial for repairing ovarian function or the biomaterial for repairing ovarian function prepared by the above-described preparation method in the preparation of products for repairing ovaries.

[0054] The inventors of this invention have discovered for the first time that the extracellular vesicles of GDF-9 overexpressed in biological materials bind to the ACVR1B receptor in ovarian cells, causing SMAD2 to phosphorylate and enter the nucleus, thereby achieving the mechanism of ovarian repair.

[0055] Extracellular vesicles themselves have a lipid bilayer structure and exhibit cytotropic effects, enabling them to bind to cells. The ACVR1B receptor on the surface of granulosa cells is a target receptor, and the modification molecule Actin A can be added to the vesicle surface. Actin A belongs to the BMP / TGF-β family and can specifically bind to ACVR1B. This allows extracellular vesicles to target ovarian granulosa cells. After the vesicle membrane engulfs and injects into the granulosa cells, GDF-9 activates the SMAD2 pathway, leading to phosphorylation, cell proliferation, and ultimately, the repair of ovarian function.

[0056] The present invention will be further illustrated by the following examples. Unless otherwise specified, the materials in the examples are prepared according to existing methods or purchased directly from the market.

[0057] Example 1

[0058] This embodiment provides a biomaterial for repairing ovarian function, which is prepared by the following method:

[0059] (a) Extract the supernatant from mesenchymal stem cells. After filtering cells and impurities using a 0.22 μm filter membrane, perform low-temperature differential centrifugation: at 4°C, centrifuge the supernatant at 330g for 10 min to remove bottom debris and retain the supernatant; centrifuge at 2000g for 10 min to remove bottom debris and retain the supernatant; remove impurities using a 0.22 μm filter membrane and retain the supernatant; centrifuge at 100000g for 85 min and retain the precipitate, which is the extracellular vesicle. Resuspend the exosomes in PBS. Add GDF-9 mRNA and Bio-Rad Gene Pulser Electroporation Buffer, mix well, place in an electroporation cuvette, and electroporate in a Lonza 4D-Nucleofector at 4°C. Allow to stand until the membrane closes to obtain GDF-9 overexpressing exosome particles.

[0060] (b) GDF-9 overexpressing extracellular vesicles were injected as the inner aqueous phase of 0.1% polyvinyl alcohol (PVA) into dichloromethane (DCM) containing PLGA as the oil phase. The entire mixture was then sonicated at 4°C. The emulsion was then immediately injected into a solution of 0.7% PVA. The secondary emulsion was emulsified on a high-speed homogenizer for 12 min to obtain the final water / oil / water (w / o / w) mixture, which is the biomaterial for repairing ovarian function.

[0061] Example 2

[0062] This embodiment provides a biomaterial for repairing ovarian function, which is prepared by the following method:

[0063] (a) Lentiviral transfection: 1. Construct the GDF-9 mRNA lentiviral vector plasmid. 2. After the mesenchymal stem cells adhere to the culture dish, add an appropriate amount of viral suspension and incubate at 37°C, changing the culture medium every 24 hours. GDF-9 overexpressing mesenchymal stem cells are obtained after 72-96 hours.

[0064] (b) GDF-9 overexpressing extracellular vesicles were injected as the inner aqueous phase of 0.1% polyvinyl alcohol (PVA) into dichloromethane (DCM) containing PLGA as the oil phase. The entire mixture was then sonicated at 4°C. The emulsion was then immediately injected into a solution of 0.7% PVA. The secondary emulsion was emulsified on a high-speed homogenizer for 12 min to obtain the final water / oil / water (w / o / w) mixture, which is the biomaterial for repairing ovarian function.

[0065] Experimental Example

[0066] Test method:

[0067] 1. Collection, purification and characterization of EVs

[0068] Human umbilical cord mesenchymal stem cells (Huc-MSCs) were cultured in medium containing 10% fetal bovine serum (FBS) in a standard cell culture incubator set at 37°C and 5% CO2. When the cells reached 80-90% confluence, they were passaged, and the cells were washed every 30 minutes with phosphate-buffered saline (PBS) to remove residual FBS. After 3 days of culture, the conditioned medium was harvested. The collected medium was then filtered through a 0.22 μm filter to remove cell debris and contaminants. The supernatant was centrifuged at 4°C, 4,000 x g for 30-60 minutes, or until the desired volume was reached, using a centrifuge with a molecular weight cutoff of 100 kDa. The concentrated EVs were washed with phosphate-buffered saline (PBS) and centrifuged at 4°C, 100,000 x g for 70 minutes to form spheroids. The supernatant was carefully discarded, and the EVS particles were resuspended in an appropriate volume of PBS or other suitable buffer. The size distribution and morphology of EVS were analyzed using transmission electron microscopy (TEM) and nanoparticle tracking analysis (TA).

[0069] 2. In vitro establishment of the DO model

[0070] Rat ovaries were extracted from animals and immediately immersed in sterile PBS containing 1% gentamicin. Subsequently, the ovaries were dissected into smaller tissue fragments and enzymatically digested in 2% collagenase IV solution for 1 hour to promote cell dissociation. The dissociation process was terminated by adding an equal volume of medium containing 20% ​​fetal bovine serum (FBS). After centrifugation, the supernatant was carefully aspirated, and the resulting cell spheres were resuspended in medium supplemented with 10% FBS. The suspension cells were then seeded into T-175 culture flasks and maintained in a standard cell culture environment at 37°C and 5% CO2. The medium was changed every 24 hours to ensure optimal cell growth and viability. After 5 days of incubation, the granulosa cell (GC) characteristics of the cultured cells were analyzed by flow cytometry. Cisplatin was administered as an inducer at the concentration determined by preliminary cell counting experiments.

[0071] 3. Total RNA sequence

[0072] Ovarian samples were obtained from DO rats and control rats and immediately immersed in cold, sterile phosphate-buffered saline (PBS) to preserve RNA integrity. Total RNA extraction was performed using TRIzol reagent (ThermoFisher Scientific, catalog number: 15596018) according to the manufacturer's instructions. Subsequently, the quality and quantity of extracted RNA were assessed using a NanoDrop ND-1000 spectrophotometer (NanoDrop, Wilmington, DE, USA) and an Agilent Bioanalyzer 2100 (Agilent Technologies, Santa Clara, CA, USA).

[0073] 4. Induction of DO model in rats

[0074] Animal care and experimental procedures strictly adhered to the guidelines established by the Animal Welfare and Ethics Committee of Beijing Hospital. A DO rat model was established using 5-7 week old female Sprague-Dawley rats. This was achieved by administering cisplatin at a dose of 3 mg / kg / day for three consecutive days. Control rats, on the other hand, received injections of physiological saline solution.

[0075] 5. Weight and ovarian reserve

[0076] Following euthanasia, the ovaries of rats in each experimental cohort were weighed to ensure consistency in subsequent analyses. After euthanasia, both ovaries were carefully dissected, and the bicornuate uterus and adipose tissue were removed to facilitate clear separation. The separated ovaries were then carefully placed on pads for standardized measurements and photographic recording. To accurately determine ovarian mass, each ovary was individually weighed using a precision balancing instrument.

[0077] 6. Follicle analysis: Histological sections of ovarian tissue were carefully examined under a microscope equipped with the scanning and analysis software SlideViewer (2.6.0).

[0078] Using established criteria from previous research by the inventors' research team (Table 1), follicles at different developmental stages were systematically classified, including primordial, secondary, and atretic follicles. Subsequently, a comprehensive follicle quantification process was performed, with three independent researchers meticulously counting the number of follicles in every ten consecutive tissue sections. Rigorous statistical analysis of the resulting dataset was conducted using GraphPad Prism software (9.5.0) to achieve robust interpretation and inference generation.

[0079] Table 1

[0080] 7. Western blots

[0081] GC cells were lysed at 4°C for 30 min using cell lysis buffer, followed by centrifugation at 15,000 rpm for 10 min. The supernatant was collected and quantified using a BCA assay kit. Equal volumes of protein were loaded onto a polyacrylamide gel for SDSPAGE separation. The separated proteins were then transferred to a polyvinylidene fluoride (PVDF) membrane. The PVDF membrane was subsequently incubated overnight at 4°C with primary antibody, followed by incubation with secondary antibody at room temperature for 1 h. The blot was visualized using an enhanced chemiluminescence assay kit.

[0082] 8. Flow cytometry

[0083] To comprehensively characterize the viability and cellular response of granulosa cells (GCs) following cisplatin-induced and extracellular vesicle (EV) treatment, flow cytometry analysis was performed using a Beckman Coulter flow cytometer. This analytical approach allows for the assessment of GC survival status, apoptosis, mechanical damage, and cell death dynamics. Specifically, the Annexin V-FITC and propidium iodide (PI) apoptosis kit was used to identify apoptotic and necrotic cell populations. This methodological framework enables the quantitative assessment of cellular responses to cisplatin-induced and subsequent EV treatment, providing important insights into the mechanisms of therapeutic interventions aimed at improving ovarian dysfunction.

[0084] 9. Immunohistochemistry and immunofluorescence

[0085] Tissue sections were dewaxed and dehydrated through a series of solvent treatments. Antigen extraction was then performed to improve immunostaining efficiency. After antigen recovery, sections were washed with PBS and incubated in 3% H2O2 PBS buffer at room temperature for 10 minutes to block endogenous peroxidase activity, followed by thorough washing. Subsequently, tissue sections were incubated overnight at 4°C with primary antibodies targeting ACVR1B, FSHR, or SMAD2. After incubation with the primary antibody, sections were washed and then incubated with the corresponding secondary antibody at room temperature for 1 hour. Visualization of the binding antibodies was achieved using 3,3'-diaminophenylbiidine (DAB) or 4',6-diamino-2-phenylindole (DAPI). Reverse staining was then performed, and quantitative analysis of the stained sections was performed using ImageJ software.

[0086] 10. Statistical Analysis

[0087] Statistical analyses were performed using GraphPad Prism software (Version 9.5). Data are presented as mean ± SD of at least three independent experiments, consistently producing comparable results. For comparisons between two groups, a two-tailed unpaired Student's t-test was used. For comparisons involving more than three groups, one-way ANOVA was performed, followed by a Bonferroni post-hoc test. Statistical significance was defined as P < 0.05.

[0088] Experimental results:

[0089] 1. Evans derived from umbilical cord mesenchymal stem cells can be separated and purified using ultrafiltration technology.

[0090] EVs are small, membrane-bound structures released from cells into the extracellular environment. EVs carry a wide variety of biomolecules, including proteins, lipids, nucleic acids, and metabolites, serving as mediators of intercellular communication and regulatory processes. In this study, the focus of this invention is the isolation and refinement of EVs from HucMSCs (Figure 2a). Using transmission electron microscopy (TEM), the morphological characteristics of the isolated EVs were carefully examined, confirming their integrity and structure (Figure 2b). Subsequent evaluation using Nano Sight helped determine the EV concentration and size, key parameters for elucidating their biophysical properties (Figure 3). Furthermore, Western blot analysis identified EV-specific markers, such as CD63 and CD9, confirming the purity and authenticity of the isolated EV population (Figure 2c). Through this systematic approach, this study meticulously characterized the properties and composition of EVs generated by HucMSCs, laying the foundation for further research into their functional roles and therapeutic potential.

[0091] 2. EVs showed the potential to enhance granulocyte viability after cisplatin-induced granulocyte injury in vitro.

[0092] To explore the functional significance of EVs in cell kinetics related to survival and apoptosis, human granulosa cells (GCs) were used as an in vitro experimental model in this study. Furthermore, a model of a dysfunctional ovary was established using cisplatin at a concentration of 2.7 μg / ml (Figure 4), which was confirmed by cell counting kit-8 (CCK8). Cells were then divided into three groups: a control group, a DO group, and an EV-treated DO group. Cell viability, apoptosis, and death were assessed in each group using flow cytometry (Figure 5). In the EV-treated DO group, the number of viable cells was significantly higher than in the DO group and approached that of the control group (Figure 6a). Furthermore, in the EV-treated DO group, the number of dead cells was significantly reduced compared to the untreated DO group, similar to the control group (Figure 6b). Moreover, no significant difference was observed in the number of mechanically damaged cells among the three groups (Figure 6c). However, the number of apoptotic cells in the EV-treated DO group was not significantly different from that in the untreated group (Figure 6d).

[0093] 3. GDF-9 plays a key role in improving GC activity and proliferation under EV stimulation.

[0094] Compared to the control group, EVs showed a significant ability to increase the number of GCs (Figure 7a and b). EVs contain multiple molecules. To investigate the target molecules in EVs for treating ovarian dysfunction (DO), this study induced GDF-9 overexpression in granulosa cells by transfecting them with GDF-9 mRNA, or inhibited GDF-9 expression by transfecting them with si-GDF-9 mRNA. After EV treatment, the number of GCs with GDF-9 overexpression was higher than that in the control group. However, EV treatment failed to increase the number of GCs when GDF-9 gene expression was inhibited (Figure 7c). Furthermore, compared to si-GDF-9 GCs treated with EVs, GDF-9 GCs showed reduced apoptosis, which was confirmed by TUNEL assays. In addition, the proliferation marker Ki-67 was expressed at a higher level in GDF-9 GCs compared to si-GDF-9 GCs (Figure 7dg).

[0095] 4. Total RNA-seq analysis showed that ACVRIB is a major regulator of GCs.

[0096] To identify target genes related to ovarian function, this study, building upon previous research, employed a cisplatin-based ovarian dysfunction model and performed total RNA sequencing analysis on normal ovaries and ovaries (Figure 8a). Differentially expressed genes were identified, including activin A receptor type 1B, which encodes a type I receptor for transforming growth factor β (TGF-β) superfamily ligands and is a key gene for cytokine-receptor interactions. KEGG pathway enrichment and pathway integration analysis revealed that upon ACVR1B phosphorylation, SMAD2 interacts with SMAD4 to form a complex (Figure 8b and c). Subsequently, this complex translocates to the nucleus, where it regulates the transcription of target genes involved in various cellular processes, such as cell proliferation, differentiation, and apoptosis.

[0097] Furthermore, to elucidate gene expression patterns under different conditions, this experimental case underwent Western blot analysis to assess the levels of GDF9, ACVR1B, and SMAD2, as well as the phosphorylation status of SMAD2. Treatment of GCs with EVs significantly increased GDF-9 protein levels. Conversely, when the GDF-9 gene was repressed by the GDF-9 antagonist follistatin, EVs failed to stimulate GDF-9 signaling. Similarly, when the GDF-9 gene was repressed, the protein expression levels of ACVR1B and SMAD2 / p-SMAD2 decreased. This observation supports the view that EVs enhance GC function through the GDF-9 / ACVR1B / SMAD2 signaling pathway (d and e in Figure 8).

[0098] Subsequently, a reverse experiment was conducted to investigate the GDF-9 / ACVR1B / SMAD2 pathway. Granulocytes (GCs) were stimulated or repressed by the GDF-9 or ACVR1B gene and then treated with EVs. Protein levels in these cells were then assessed by Western blot analysis. Gene manipulation targeting GDF-9 and ACVR1B was performed on GCs, followed by reintroduction of the functional genes via repair constructs. Notably, reactivation of the ACVR1B-mediated signaling pathway was observed after restoration of GDF-9 expression in GCs, as evidenced by phosphorylation of the downstream effector SMAD2. When the ACVR1B gene was stimulated in GCs with ALK-4 but the GDF-9 gene was knocked out, a significant loss of SMAD2 protein expression was observed in GCs. Knockout of ACVR1B in granulocytes (GCs) using the inhibitor SB431542 resulted in the inability of GCs to express the specific protein GDF-9 (f and g in Figure 8). This observation highlights the crucial role of ACVR1B in regulating GDF-9 expression in GCs, emphasizing its importance in mediating granulocyte function and reproductive processes. This compelling evidence underscores the hierarchical relationship within the GDF-9 / ACVR1B / SMAD2 pathway, confirming that GDF-9 is an upstream regulator in initiating the ACVR1B-mediated signaling cascade in GCs.

[0099] 5. Upregulation of GDF-9 by EVs can restore ovarian function in rats.

[0100] Stimulating the GDF-9 gene in HucMSCs and then isolating GDF-9-overexpressing extracellular vesicles (GOEs) provides a promising approach for therapeutic intervention in a cisplatin-induced ovarian dysfunction (DO) rat model. To comprehensively investigate the effects of GOEs on ovarian dysfunction, a controlled animal study was designed in this experiment. Three distinct groups were established: a control group, a DO group, and a GOE-treated DO group (Figure 9). After administration of purified GOEs to DO rats, body weight, ovarian size, and weight were significantly increased compared to untreated DO rats (ad in Figures 10 and 11). Furthermore, histological examination showed a significant increase in the number of primary follicles (PMF) and secondary follicles (SCF) in the GOE-treated group, while the number of atretic follicles (ATF) was significantly reduced (eh in Figure 11). These compelling findings collectively suggest that GOEs have a multifaceted mechanism of action, including promoting follicular development, inhibiting follicular atresia, and enhancing overall ovarian function. This potent therapeutic effect underscores the potential utility of GOEs as a novel and promising treatment approach to alleviate ovarian dysfunction and restore ovarian health characteristic of DO. This case study provides valuable insights into the therapeutic potential of GOEs and highlights their promising role in addressing ovarian dysfunction, thus offering new avenues for developing targeted interventions aimed at improving reproductive health outcomes in affected individuals.

[0101] 6. GOEs may enhance ovarian function by stimulating the expression of ACVRIB in GCs.

[0102] Furthermore, this study employed a combination of immunohistochemistry (IHC) and immunofluorescence (IF) analysis to investigate the effects of GOEs on ACVR1B expression in DO GCs. The use of IHC and IF techniques provided a comprehensive assessment of ACVR1B expression in GCs, offering complementary perspectives on the observed effects (Figure 13a, b). Results from both methods consistently showed a significant upregulation of ACVR1B expression in GCs of both the control and GOE-treated DO ovaries compared to untreated DO ovaries (Figure 13c, d). This strong evidence powerfully confirms the hypothesis that GOE treatment enhances ACVR1B expression in GCs of DO ovaries, thus outlining a promising therapeutic trajectory for alleviating ovarian dysfunction. By employing this multifaceted approach, this study not only enhanced the validity and reliability of the results but also provided a deeper understanding of the complex molecular mechanisms underlying GOE-mediated regulation of ACVR1B expression within the physiological and pathological context of the ovary.

[0103] Furthermore, as shown in Figure 12, to improve the accuracy and specificity of this study, the follicle-stimulating hormone receptor (FSHR) was integrated as a marker to distinguish GCs within the ovarian tissue microenvironment. FSHR is a key receptor expressed on the surface of GCs, coordinating crucial processes related to follicular development and ovulation. Using FSHR as a recognition marker facilitates the precise identification and visualization of GCs within ovarian tissue sections, enabling detailed characterization of ACVR1B expression dynamics within this cellular environment. This strategic integration of FSHR not only optimizes the accuracy and resolution of this study but also provides valuable insights into the complex molecular crosstalk supporting the effects of GOEs on ovarian function. Through this meticulous and comprehensive approach, this study provides compelling evidence depicting the crucial role of GOEs in regulating ACVR1B expression within GCs, paving the way for the development of targeted therapeutic interventions aimed at improving ovarian dysfunction and promoting reproductive health outcomes.

[0104] 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 or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A biomaterial for restoring ovarian function, characterized by, This includes extracellular vesicles that overexpress GDF-9 as active substances.

2. The biomaterial of claim 1, wherein, The biomaterial is a membrane-type agent; Preferably, the biomaterial is in tablet, powder, emulsion, or liquid form.

3. The method for preparing a biological material for restoring ovarian function according to claim 1 or 2, characterized in that, Includes the following steps: (a) Preparation of extracellular vesicles overexpressing GDF-9; (b) The GDF-9 overexpressing extracellular vesicles obtained in step (a) are prepared as the biomaterial for repairing ovarian function.

4. The production method according to claim 3, characterized by, Step (a) includes inducing GDF-9 overexpression in mesenchymal stem cells with GDF-9 mRNA, collecting cell supernatant, and obtaining GDF-9 overexpressing extracellular vesicles.

5. The preparation method according to claim 4, characterized in that, The sequence of the GDF-9 mRNA is Homo sapiens growth differentiation factor 9 (GDF9), RefSeqGene on chromosome 5, Sequence ID: NG_047051.1:5416-10700.

6. The method of any one of claims 3-5, wherein, The methods for inducing GDF-9 overexpression in mesenchymal stem cells are selected from cell transfection or electroporation.

7. The preparation method according to claim 3, characterized in that, Step (b) includes injecting the GDF-9 overexpressing extracellular vesicles prepared in step (a) as the inner aqueous phase of 0.1% polyvinyl alcohol into dichloromethane containing PLGA as the oil phase, sonicating them, and then injecting them into a solution of 0.7% PVA. After emulsification, the biomaterial for repairing ovarian function is obtained.

8. The method of claim 7, wherein, Before step (b), the method further includes the step of preparing the overexpressing GDF-9 extracellular vesicles into a lyophilized powder.

9. The application of the biomaterial for repairing ovarian function as described in claim 1 or 2, or the biomaterial for repairing ovarian function prepared by any one of the preparation methods described in claims 3-8, in the preparation of products for repairing ovaries.

10. Use according to claim 9, characterized in that, The overexpression of GDF-9 in the biomaterials causes extracellular vesicles to bind to the ACVR1B receptor in ovarian cells, leading to phosphorylation of SMAD2 into the nucleus and thus repairing the ovary.

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