Use of a collagen membrane complex loaded with stem cells in the preparation of a material for cervical regeneration

Abstract

This disclosure provides the application of a collagen membrane complex loaded with stem cells in the preparation of materials for cervical regeneration. The preparation method of the collagen membrane complex loaded with stem cells includes: S1, digesting stem cells with trypsin to prepare a single-cell suspension; S2, adding the single-cell suspension dropwise onto the surface of a collagen membrane and culturing in an incubator; S3, adding culture medium and continuing culturing to form a collagen membrane complex loaded with stem cells. The collagen membrane complex loaded with stem cells, with a bilayer structure, prepared using this method is beneficial for cervical regeneration.

Description

Technical Field

[0001] This disclosure relates to the field of obstetrics and gynecology, and more specifically, to the use of a collagen membrane complex loaded with stem cells in the preparation of materials for cervical regeneration. Background Technology

[0002] Vaginal atresia refers to a condition where the ovaries and endometrium are normally developed, but the vagina is completely or partially replaced by fibrous tissue. Type I vaginal atresia is characterized by lower vaginal atresia with a normally developed upper vaginal segment and cervix; Type II vaginal atresia is characterized by complete vaginal atresia, combined with complete or partial cervical hypoplasia. Due to the complete vaginal atresia and cervical hypoplasia, Type II vaginal atresia makes it easier for menstrual blood to reflux into the pelvic cavity, increasing the risk of endometriosis. Because patients lack normal cervical tissue, previous cervicoplasty surgeries required long-term placement of cervical drainage tubes to support and drain menstrual blood, which easily leads to complications such as cervical restenosis and pelvic infection. In severe cases, it can cause sepsis and death, seriously affecting the patient's health. There is currently no method to regenerate tissue. Therefore, cervical tissue regeneration is crucial to overcoming the shortcomings of current cervicoplasty procedures.

[0003] Stem cells possess immense potential for tissue regeneration and repair, and numerous clinical studies have confirmed their therapeutic effects in vivo. Mesenchymal stem cells (MSCs), for example, exhibit strong self-renewal and differentiation potential, capable of differentiating into specific cell types such as smooth muscle cells and fibroblasts. Simultaneously, MSCs can also promote tissue regeneration by improving the tissue microenvironment through paracrine effects. Currently, there are animal studies reporting the use of MSCs for cartilage injury repair and vaginal repair. However, directly applying stem cells to in vivo treatment presents challenges such as difficulty in local localization and the inability to maintain cell viability and stemness long-term, thus affecting efficacy.

[0004] Collagen is a major component of the extracellular matrix and has advantages such as good biocompatibility and biodegradability. Collagen scaffolds have been approved for clinical use in skin and oral mucosal repair. Previous reports have shown that vitrified collagen solutions encapsulating nylon sutures can provide mechanical dilation at cervical stenosis sites in rabbits, preventing cervical stenosis and scar fibrosis. However, there are no reports on stem cell composite collagen membrane scaffolds in cervical regeneration and cervical tissue repair. Summary of the Invention

[0005] The purpose of this disclosure is to overcome the shortcomings of existing technologies that cannot regenerate cervical tissue, and to provide an application of a collagen membrane complex loaded with stem cells in the preparation of materials for cervical regeneration, providing a material and method for tissue regeneration for cervical hypoplasia or postoperative cervical loss.

[0006] To achieve the above objectives, this disclosure provides the application of a collagen membrane complex loaded with stem cells in the preparation of materials for cervical regeneration, wherein the collagen membrane complex loaded with stem cells is prepared by a method comprising the following steps:

[0007] S1. Stem cells were digested with trypsin and then prepared into a single-cell suspension.

[0008] S2. The single-cell suspension is dropped onto the surface of the collagen membrane and placed in an incubator for incubation.

[0009] S3. Add culture medium and continue culturing to form a collagen membrane complex loaded with stem cells.

[0010] According to this disclosure, the collagen membrane is a collagen membrane obtained by decellularizing pig skin and has a double-layer structure; the length of the collagen membrane is 5-200 mm, the width is 5-100 mm, and the thickness of each layer of the collagen membrane is 0.1-10 mm.

[0011] According to this disclosure, the surface of the collagen membrane bilayer structure includes a rough surface and a smooth surface, wherein, in step S2, the single-cell suspension is drop-added to the rough surface of the collagen membrane.

[0012] According to this disclosure, in step S2, the seeding amount of the collagen membrane surface stem cells is 10. 4 -10 7 pcs / cm 2 Preferably 4×10 5 pcs / cm 2 .

[0013] According to this disclosure, the stem cells are selected from one or more of embryonic stem cells, pluripotent induced stem cells, adult stem cells and mesenchymal stem cells;

[0014] The mesenchymal stem cells are selected from one or more of bone marrow mesenchymal stem cells, adipose mesenchymal stem cells, umbilical cord mesenchymal stem cells, and uterine blood mesenchymal stem cells.

[0015] According to this disclosure, the culture conditions in the incubator can be the conventional culture conditions in the cell culture process, preferably 37°C, 5% CO2, and the culture time is 1-7 days.

[0016] According to this disclosure, the culture medium includes at least one of stem cell culture medium, differentiation culture medium, and DEME culture medium.

[0017] Through the above technical solution, this disclosure loads stem cells onto the surface of a collagen membrane to form a collagen membrane complex for preparing cervical regeneration materials. In this collagen membrane complex, stem cells, as the active ingredient for treating cervical dysplasia, possess strong self-renewal and differentiation potential, capable of differentiating into specific cell types of cervical tissue, promoting regeneration, and can also improve the tissue microenvironment through paracrine effects to promote tissue regeneration. The collagen membrane, as a placement material after cervicoplasty, has a bilayer structure, good biocompatibility, and a mechanical expansion effect on cervical tissue to prevent restenosis; simultaneously, it can serve as a support site for stem cells, maintaining their cell activity, stemness, and duration of action; furthermore, the collagen membrane is degradable and does not require secondary removal. Therefore, this disclosure provides a treatment method for cervical regeneration and further improves the therapeutic effect of cervical regeneration.

[0018] Other features and advantages of this disclosure will be described in detail in the following detailed description section. Attached Figure Description

[0019] 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:

[0020] Figure 1 This is an electron microscope image of the rough / smooth surface of the collagen membrane disclosed in this publication.

[0021] Figure 2 This shows the growth state of MSCs on the collagen membrane in the collagen membrane complex prepared in this disclosure (the blocky structure in the figure represents live cells, with no dead cells).

[0022] Figure 3 This is a comparison image of the repair effect of huMSCs collagen membrane complex on full-thickness cervical damage in rabbits (left: before treatment; right: after treatment). Detailed Implementation

[0023] 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.

[0024] This disclosure provides the application of a collagen membrane complex loaded with stem cells in the preparation of materials for cervical regeneration, wherein the collagen membrane complex loaded with stem cells is prepared by a method comprising the following steps:

[0025] S1. Stem cells were digested with trypsin and then prepared into a single-cell suspension.

[0026] S2. The single-cell suspension is dropped onto the surface of the collagen membrane and placed in an incubator for incubation.

[0027] S3. Add culture medium and continue culturing to form a collagen membrane complex loaded with stem cells.

[0028] Optionally, the collagen membrane is a collagen membrane obtained by decellularizing pig skin and has a double-layer structure; the length of the collagen membrane is 5-200 mm, the width is 5-100 mm, and the thickness of each layer is 0.1-10 mm.

[0029] Optionally, the surface of the collagen membrane bilayer structure includes a rough surface and a smooth surface, and the single-cell suspension is dropped onto the rough surface of the collagen membrane.

[0030] The collagen membrane disclosed herein has a bilayer structure, and its internal structure and the thickness of each layer are suitable for tissue remodeling. It can meet the growth needs of natural tissue cells, provide support sites for tissue regeneration, and maintain its cell activity, stemness, and duration of action.

[0031] Optionally, in step S2, the seeding amount of stem cells on the collagen membrane surface is 10. 4 -10 7 pcs / cm 2 Preferably 4×10 5 pcs / cm 2 .

[0032] The stem cells disclosed herein can secrete growth factors, providing active ingredients for tissue regeneration, and can also improve the local microenvironment through paracrine function, thereby promoting tissue regeneration.

[0033] Optionally, the stem cells are selected from one or more of embryonic stem cells, pluripotent induced stem cells, adult stem cells, and mesenchymal stem cells; preferably, the stem cells are mesenchymal stem cells.

[0034] The mesenchymal stem cells are selected from one or more of bone marrow mesenchymal stem cells, adipose mesenchymal stem cells, umbilical cord mesenchymal stem cells, and uterine blood mesenchymal stem cells.

[0035] Optionally, in step S2, the single-cell suspension is dropped onto the surface of the collagen membrane and placed in an incubator under conventional cell culture conditions, preferably 37°C, 5% CO2, for 1-7 days.

[0036] Optionally, the culture medium includes at least one of stem cell culture medium, differentiation culture medium, and DEME culture medium.

[0037] The present disclosure is further described in detail below through examples.

[0038] Example 1

[0039] Stem cell culture: Steps for isolating, purifying, and culturing huMSCs (human umbilical cord mesenchymal stem cells): Collect fresh umbilical cord, remove surface blood, and cut into small tissue blocks of approximately 0.5 cm using scissors; add trypsin and digest in an incubator for 30 minutes, observing the degree of cell release under a microscope; aspirate the digestion supernatant, seed the digested tissue blocks into culture flasks with approximately 1 cm intervals between each block, add stem cell culture medium, and culture for 2-3 days to allow the tissue blocks to fully adhere to the culture flask; change the culture medium every 2-3 days and observe the cell growth from the tissue blocks; remove the tissue blocks for cryopreservation, and passage culture when the cells have reached 70% confluence with the culture flask.

[0040] Cell seeding process: Steps for co-culturing huMSCs with collagen membranes: (The collagen membrane is obtained by decellularizing porcine skin tissue to obtain a bilayer structure, such as...) Figure 1 As shown, one layer has a smooth surface and the other layer has a rough surface. The length is 20 mm, the thickness is 3 mm, and the width is 10 mm. (1) Culture MSCs to 80% density (i.e., the end of the exponential growth phase); (2) Prepare a cell suspension after trypsin digestion, count the cells and adjust the cell density to 2 × 10⁻⁶. 6 (3) Soak the collagen membrane in DMEM culture medium overnight, and the next day use sterile filter paper or sterile gauze to absorb the liquid in the collagen membrane; (4) Add the MSCs cell suspension evenly to the rough surface of the moistened collagen membrane. The cell suspension will automatically be adsorbed into the collagen membrane, per cm 2 (5) Add 100 μL of cell suspension to the collagen membrane material of the area; (6) Place the collagen membrane loaded with stem cells in a culture dish, and then place it in a cell culture incubator for 4 hours to allow the cells to adhere; (7) After cell adhesion, gently wash away the unadsorbed stem cells with PBS, add complete culture medium, and culture for 5 days to form a collagen membrane complex loaded with stem cells. The growth status of MSCs on the collagen membrane matrix material is as follows: Figure 2 As shown ( Figure 2 The blocky structure represents living cells, with no dead cells present.

[0041] Example 2

[0042] A rabbit cervical full-thickness injury repair experiment using a collagen membrane complex loaded with MSCs:

[0043] (1) Control group: The right cervix of the rabbit was completely removed and a urinary catheter was inserted; two sutures were inserted 2cm into the uterus to fix the uterus and the urinary catheter, and the excess part of the urinary catheter that protruded outward was cut off;

[0044] (2) MSCs composite material group: The right cervix of the rabbit was completely removed and the urinary catheter wrapped with a collagen membrane complex loaded with stem cells was sutured end to end.

[0045] The collagen membrane complex loaded with MSCs was transplanted into a rabbit model of cervical dysplasia:

[0046] (1) After anesthetizing and shaving the rabbits at 0.1 mL / kg body weight, the skin and peritoneum were cut open and the bladder was everted to expose the bladder. The urine was drained with a syringe to expose the vagina and the two uteri. (2) The 2*1 cm collagen membrane complex loaded with MSCs prepared as in Example 1 was sutured into a column along the long axis. (3) The cervix was cut flush with the full thickness and the cut tissue was discarded. The sutured collagen membrane complex loaded with stem cells was placed in contact with the cut cervix along the short axis. The outer layer material was sutured to the outer layer of the cervix with simple interrupted sutures. The inner layer material was sutured to the inner layer of the cervix with simple interrupted sutures. The cervixes were everted into the vagina and the vaginal wall was sutured with 6-0 absorbable sutures. The peritoneum and skin were sutured with 3-0 non-absorbable sutures.

[0047] The results are as follows Figure 3 As shown, collagen membrane complex loaded with MSCs stem cells can promote cervical regeneration. In the left image, the untreated cervix showed no tissue regeneration; in the right image, after treatment with collagen membrane complex loaded with stem cells, the cervix can regenerate to the same height as the normal cervix on the opposite side.

[0048] The method for preparing the collagen membrane complex loaded with stem cells disclosed herein is simple, low-cost, and can effectively promote cervical regeneration.

[0049] 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.

[0050] 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.

[0051] 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 stem cells in the preparation of materials for cervical regeneration, characterized in that, The collagen membrane complex loaded with stem cells was prepared by a method comprising the following steps: S1. Stem cells were digested with trypsin and then prepared into a single-cell suspension. S2. The single-cell suspension is dropped onto the surface of the collagen membrane and placed in an incubator for incubation. S3. Add culture medium and continue culturing to form a collagen membrane complex loaded with stem cells; The collagen membrane has a bilayer structure; the surfaces of the bilayer structure of the collagen membrane include a rough surface and a smooth surface. In step S2, the single-cell suspension is dropwise added to the rough surface of the collagen membrane; the seeding amount of stem cells on the collagen membrane surface is 10. 4 -10 7 pcs / cm 2 ; The collagen membrane has a length of 5-200mm, a width of 5-100mm, and a thickness of 0.1-10mm for each layer. The collagen membrane is a collagen membrane obtained by decellularizing pig skin.

2. The application according to claim 1, wherein, The collagen membrane surface stem cells are 4×10 5 pcs / cm 2 .

3. The application according to claim 1, wherein, The stem cells are selected from one or more of embryonic stem cells, pluripotent induced stem cells, adult stem cells and mesenchymal stem cells; The mesenchymal stem cells are selected from one or more of bone marrow mesenchymal stem cells, adipose mesenchymal stem cells, umbilical cord mesenchymal stem cells, and uterine blood mesenchymal stem cells.

4. The application according to claim 1, wherein, The culture medium includes at least one of stem cell culture medium, differentiation culture medium, and DEME culture medium.

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