Use of a collagen membrane complex loaded with exosomes in the preparation of a material for cervical repair

Abstract

The present application provides a use of an exosome-loaded collagen membrane complex in the preparation of a material for cervical repair, and a preparation method of the exosome-loaded collagen membrane complex includes fusion culture of exosomes and a collagen membrane. The exosome-loaded collagen membrane complex can quickly promote epithelial tissue regeneration, thereby preventing cervical restenosis, and the exosome-loaded collagen membrane has a double-layer structure, which is beneficial to the growth of different types of cells.

Description

Technical Field

[0001] This disclosure relates to the field of tissue repair, and more specifically, to the use of a collagen membrane complex loaded with exosomes in the preparation of materials for cervical repair. Background Technology

[0002] Congenital cervical dysplasia is a rare disease of the female reproductive system, often accompanied by vaginal dysplasia. Because menstrual blood cannot be discharged, it severely impacts the patient's physical and mental health. The main treatment involves incising the closed lower uterine segment and anastomosing the opening between the rectum and vesicourethra to allow menstrual blood to drain. The biggest problem with this surgery is the high chance of re-adhesion and closure due to the absence of a normal cervix and mucosa. Therefore, cervical and mucosal regeneration remains a major challenge, and there is still no effective treatment.

[0003] Mesenchymal stem cell exosomes are key components of paracrine signaling in mesenchymal stem cells. With a particle size of approximately 40-150 nm, they encapsulate bioactive molecules such as proteins, mRNA, and miRNA, which play a role in signal transduction and possess functions similar to those of mesenchymal stem cells in promoting the growth, differentiation, and repair of corresponding cells. Exosomes also exhibit advantages such as high stability, low immunogenicity, and ease of storage, making them highly promising for tissue regeneration applications. However, due to their rapid elimination from the body and relatively short in vivo half-life, the current delivery methods for exosomes remain a major challenge, as existing methods cannot achieve concentrated and long-term release of exosomes at the treatment site.

[0004] Therefore, there is an urgent need to develop a biocompatible material that can maintain the activity and continuous release of exosomes to achieve targeted and sustained release of exosomes on wound surfaces, and to apply it to cervical repair to achieve rapid cervical epithelial growth, prevent cervical restenosis, and promote cervical regeneration. Summary of the Invention

[0005] The purpose of this disclosure is to provide the application of an exosome-loaded collagen membrane complex in the preparation of materials for cervical repair. This exosome-loaded collagen membrane complex has a bilayer structure, which is beneficial for the growth of different cell types, enabling rapid epithelialization, effective prevention of cervical restenosis, and promotion of cervical regeneration.

[0006] To achieve the above objectives, this disclosure provides the application of an exosome-loaded collagen membrane complex in the preparation of materials for cervical repair, wherein the preparation method of the exosome-loaded collagen membrane complex includes fusing and culturing exosomes with a collagen membrane.

[0007] Optionally, the exosomes are extracted from body fluids or cells;

[0008] The bodily fluids include at least one of blood, saliva, urine, breast milk, cerebrospinal fluid, ascites, and amniotic fluid;

[0009] The cells include at least one of stem cells, fibroblasts, epithelial cells, and mesenchymal cells, preferably umbilical cord mesenchymal stem cells.

[0010] Optionally, the collagen membrane has a double-layer structure, the outer surface of the collagen membrane includes a rough surface and a smooth surface, the length of the collagen membrane is 5-200mm, the width is 5-100mm, and the thickness of each layer of the collagen membrane is 0.5-10mm.

[0011] Optionally, the inoculation amount of exosomes on the collagen membrane surface is 10. 5 -10 15 pcs / cm 2 Preferably 3×10 10 pcs / cm 2 .

[0012] Optionally, the fusion culture process includes: dropping the exosomes onto the rough surface of the collagen membrane, and obtaining the collagen membrane complex loaded with exosomes after full wetting.

[0013] Optionally, the cervical repair includes preventing cervical restenosis, promoting rapid epithelialization, and promoting cervical regeneration.

[0014] Through the above technical solution, this disclosure uniformly distributes exosomes within a collagen membrane to form an exosome-loaded collagen membrane complex with directional release function. Using this exosome-loaded collagen membrane complex in the preparation of materials for cervical repair can achieve several beneficial effects: it enables targeted sustained-release therapy, treats cervical epithelial damage, and promotes epithelial regeneration; the collagen membrane and exosomes have good compatibility and stable binding, exhibiting superior regulatory effects at specific treatment sites, enhancing the local effects of exosomes, and better promoting tissue regeneration and repair.

[0015] Other features and advantages of this disclosure will be described in detail in the following detailed description section. Attached Figure Description

[0016] The accompanying drawings are provided to further illustrate the present disclosure and form part of the specification. They are used together with the following detailed description to explain the present disclosure, but do not constitute a limitation thereof. In the drawings:

[0017] Figure 1 This is an electron microscope image of MSC-Exo.

[0018] Figure 2 This is an electron microscope image of the collagen membrane surface.

[0019] Figure 3 This is an IF image of MSC-Exo binding to the collagen membrane.

[0020] Figure 4 This is the MSC-Exo release curve after MSC-Exo binds to the collagen membrane.

[0021] Figure 5 These are before-and-after comparison images of the repair of rabbit cervical epithelial damage by collagen membrane complex. Detailed Implementation

[0022] The specific embodiments of this disclosure will be described in detail below with reference to the accompanying drawings. It should be understood that the specific embodiments described herein are for illustration and explanation only and are not intended to limit this disclosure.

[0023] This disclosure provides the application of an exosome-loaded collagen membrane complex in the preparation of materials for cervical repair, wherein the preparation method of the exosome-loaded collagen membrane complex includes fusing and culturing exosomes with a collagen membrane.

[0024] Optionally, cervical repair includes preventing cervical restenosis, promoting rapid epithelialization, and promoting cervical regeneration.

[0025] Optionally, the exosomes are extracted from body fluids or cells.

[0026] Optionally, the body fluid includes at least one of blood, saliva, urine, breast milk, cerebrospinal fluid, ascites, and amniotic fluid;

[0027] Optionally, the cells include at least one of stem cells, fibroblasts, epithelial cells, and mesenchymal cells, preferably umbilical cord mesenchymal stem cells.

[0028] Optionally, the collagen membrane has a double-layer structure, and the outer surface of the collagen membrane includes a rough surface and a smooth surface; the length of the collagen membrane is 5-200mm, the width is 5-100mm, and the thickness of each layer of the collagen membrane is 0.5-10mm.

[0029] Optionally, the inoculation amount of exosomes on the collagen membrane surface is 10. 5 -10 15 pcs / cm 2 Preferably 3×10 10 pcs / cm 2 .

[0030] Optionally, the fusion culture process includes: dropping the exosomes onto the rough surface of the collagen membrane, and obtaining the collagen membrane complex loaded with exosomes after full wetting.

[0031] The present disclosure is further described in detail below through examples.

[0032] Example 1

[0033] Exosome isolation and identification: P3-P6 generation human umbilical cord mesenchymal stem cells were cultured at 80-90% confluence density after cell fusion. The culture medium was replaced with exosome-free serum and cultured for 48 hours. The cell culture medium was collected. The cells were then subjected to a gradient centrifugation: 300×g for 10 min at 4°C, and the supernatant was collected; centrifuged at 2000×g for 20 min at 4°C; centrifuged at 10000×g for 30 min at 4°C; and ultracentrifuged at 100,000×g for 70 min at 4°C. The precipitate was resuspended in PBS and ultracentrifuged again at 100,000×g for 70 min at 4°C. The supernatant was discarded, and the precipitate was the exosomes. After filtration through a 0.22 mm filter membrane, the cells were stored at -80°C. Electron micrographs of the umbilical cord mesenchymal stem cells are shown below. Figure 1 As shown, the exosomes were observed to be round vesicles with a diameter of 40-100 nm, and the exosomes MSC-Exo of human umbilical cord mesenchymal stem cells were successfully isolated.

[0034] Collagen membrane material: The collagen membrane has a double-layer structure with a smooth surface and a rough surface, with a length of 20mm, a thickness of 3mm, and a width of 10mm.

[0035] Composite MSC-Exo and collagen membrane material: 200 μL concentration of 3 × 10 11 MSC-Exo cells / mL were added dropwise to a volume of 2.0 × 1.0 cm. 2 On the rough surface of the collagen membrane (rough surface such as...) Figure 2 As shown in the figure, allow the MSC-Exo solution to fully wet the material.

[0036] Release assay: Collagen membranes loaded with exosomes were immersed in 100 μL of PBS; 100 μL of the precipitate was collected every 24 hours, and fresh 100 μL of PBS was added each time for 14 consecutive days. The concentration of MSC-Exo was measured by NTA. The experiment was repeated three times. The if diagram of the collagen membrane loaded with MSC-Exo is shown in the figure. Figure 3 As shown, exosomes were successfully loaded onto the collagen membrane, and the dots in the figure represent MSC-Exo. The MSC-Exo release curve is shown below. Figure 4 As shown, MSC-Exo is slowly released onto the collagen membrane for up to 14 days, indicating good compatibility between MSC-Exo and the collagen membrane.

[0037] Experiment on the promotion of mucosal epithelial regeneration by loading MSC-Exo collagen membrane: MSC-Exo composite collagen membrane was implanted into a rabbit cervical epithelial injury model. After euthanasia on day 90 post-operation, tissue samples were harvested for histological examination to observe the appearance of the regenerated cervix. Results are as follows: Figure 5 As shown, the left image is the control group, where the side of the cervical endometrium damaged and untreated showed stenosis leading to hydronephrosis; the right image is the treatment group, where the cervix and uterus appeared normal with no obvious hydronephrosis.

[0038] The preferred embodiments of this disclosure have been described in detail above with reference to the accompanying drawings. However, this disclosure is not limited to the specific details of the above embodiments. Within the scope of the technical concept of this disclosure, various simple modifications can be made to the technical solutions of this disclosure, and these simple modifications all fall within the protection scope of this disclosure.

[0039] It should also be noted that the various specific technical features described in the above embodiments can be combined in any suitable manner without contradiction. To avoid unnecessary repetition, this disclosure will not describe the various possible combinations separately.

[0040] Furthermore, various different embodiments of this disclosure can be combined in any way, as long as they do not violate the spirit of this disclosure, they should also be regarded as the content disclosed in this disclosure.

Claims

1. The application of a collagen membrane complex loaded with exosomes in the preparation of materials for cervical repair, characterized in that, The method for preparing the collagen membrane complex loaded with exosomes includes fusing and culturing exosomes with a collagen membrane; The collagen membrane has a double-layer structure; the outer surface of the collagen membrane includes a rough surface and a smooth surface. The fusion culture process includes: dropping the exosomes onto the rough surface of the collagen membrane, and obtaining the collagen membrane complex loaded with exosomes after full wetting; The collagen membrane has a length of 5-200mm, a width of 5-100mm, and a thickness of 0.5-10mm for each layer. The inoculation amount of the exosomes on the collagen membrane surface was 10. 5 -10 15 pcs / cm 2 .

2. The application according to claim 1, wherein, The exosomes are extracted from body fluids or cells; The bodily fluids include at least one of blood, saliva, urine, breast milk, cerebrospinal fluid, ascites, and amniotic fluid; The cells include at least one of stem cells, fibroblasts, epithelial cells, and mesenchymal cells.

3. The application according to claim 2, wherein, The cells in question are umbilical cord mesenchymal stem cells.

4. The application according to claim 1, wherein, The inoculation amount of exosomes on the collagen membrane surface was 3 × 10⁻⁶. 10 pcs / cm 2 .

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