CN116459397A - Application of collagen membrane compound loaded with stem cells in preparation of material for cervical regeneration - Google Patents
Application of collagen membrane compound loaded with stem cells in preparation of material for cervical regeneration Download PDFInfo
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- CN116459397A CN116459397A CN202310301363.8A CN202310301363A CN116459397A CN 116459397 A CN116459397 A CN 116459397A CN 202310301363 A CN202310301363 A CN 202310301363A CN 116459397 A CN116459397 A CN 116459397A
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- A—HUMAN NECESSITIES
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- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
- A61L27/38—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells
- A61L27/3804—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells characterised by specific cells or progenitors thereof, e.g. fibroblasts, connective tissue cells, kidney cells
- A61L27/3834—Cells able to produce different cell types, e.g. hematopoietic stem cells, mesenchymal stem cells, marrow stromal cells, embryonic stem cells
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- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/14—Macromolecular materials
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- A61L27/24—Collagen
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
- A61L27/3683—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix subjected to a specific treatment prior to implantation, e.g. decellularising, demineralising, grinding, cellular disruption/non-collagenous protein removal, anti-calcification, crosslinking, supercritical fluid extraction, enzyme treatment
- A61L27/3687—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix subjected to a specific treatment prior to implantation, e.g. decellularising, demineralising, grinding, cellular disruption/non-collagenous protein removal, anti-calcification, crosslinking, supercritical fluid extraction, enzyme treatment characterised by the use of chemical agents in the treatment, e.g. specific enzymes, detergents, capping agents, crosslinkers, anticalcification agents
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- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
- A61L27/38—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells
- A61L27/3895—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells using specific culture conditions, e.g. stimulating differentiation of stem cells, pulsatile flow conditions
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- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
- A61L27/58—Materials at least partially resorbable by the body
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- A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
- A61L2300/20—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices containing or releasing organic materials
- A61L2300/30—Compounds of undetermined constitution extracted from natural sources, e.g. Aloe Vera
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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- A61L2430/00—Materials or treatment for tissue regeneration
- A61L2430/22—Materials or treatment for tissue regeneration for reconstruction of hollow organs, e.g. bladder, esophagus, urether, uterus
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Abstract
The present disclosure provides an application of a collagen membrane complex loaded with stem cells in preparing a material for cervical regeneration, and a preparation method of the collagen membrane complex loaded with stem cells includes: s1, preparing single-cell suspension after stem cell pancreatin digestion; s2, dripping the single-cell suspension liquid onto the surface of a collagen membrane, and placing the collagen membrane into an incubator for culture; and S3, adding a culture medium for continuous culture to form a collagen membrane compound loaded with stem cells. The collagen membrane compound with the double-layer structure and the load of the stem cells prepared by the method is beneficial to cervical regeneration.
Description
Technical Field
The present disclosure relates to the field of obstetrics and gynecology, in particular, to the use of a stem cell loaded collagen membrane composite in the preparation of a material for cervical regeneration.
Background
Vaginal occlusion refers to the normal development of the patient's ovaries, endometrium, but the vagina is completely or partially replaced by fibrous tissue. The type I vaginal occlusion is an occlusion of the lower vaginal segment, and comprises an upper vaginal segment and a cervical which are normal in development; type II vaginal locks are manifested as complete vaginal locks, combined with complete or partial cervical dysplasia. Type II vaginal occlusion has increased chances of endometriosis due to complete vaginal occlusion and cervical dysplasia, with menstrual blood easily flowing back to the pelvic cavity. Since the patient has no normal cervical tissue, the cervical drainage tube needs to be placed for a long time to support and exclude menstrual blood after the past operation cervical plasty, complications such as cervical restenosis and pelvic infection are easy to occur, severe cases can cause septicemia to cause death, the health of the patient is seriously affected, and no method for regenerating the tissue exists. Therefore, regeneration of cervical tissue in patients is critical to address the deficiencies of current cervical angioplasty.
Stem cells have great potential in tissue regeneration and repair, and several clinical studies have also demonstrated their therapeutic effects in vivo. Taking mesenchymal stem cells (mesenchymal stem cells, MSCs) as an example, they have strong self-renewal and differentiation potential, and can differentiate into specific cell types such as smooth muscle cells and fibroblasts, and at the same time, the mesenchymal stem cells can also improve the tissue microenvironment by paracrine action to promote tissue regeneration. At present, animal experiments for repairing cartilage injury and vagina by using mesenchymal stem cells are reported, but when the mesenchymal stem cells are directly used for in vivo treatment, the mesenchymal stem cells have the defects that local positioning is difficult, cell activity and cell stem property cannot be maintained for a long time, and the curative effect is affected.
Collagen is the main component of extracellular matrix, has the advantages of good biocompatibility, degradability and the like, and the collagen scaffold is approved to be applied to clinical skin and oral mucosa repair. The vitrified collagen solution Bao Bei nylon wire can play a role in mechanical expansion at the cervical stenosis part of the rabbit to prevent cervical stenosis and scar fibrosis, but no report on regeneration of the cervix and repair of cervical tissues by the stem cell composite collagen membrane scaffold is available.
Disclosure of Invention
The invention aims to overcome the defect that cervical tissues cannot be regenerated in the prior art, and provides an application of a collagen membrane compound loaded with stem cells in preparing a material for cervical regeneration, and a material and a method for tissue regeneration for cervical dysplasia or postoperative cervical loss.
In order to achieve the above object, the present disclosure provides a use of a stem cell-loaded collagen membrane composite prepared by a method comprising the steps of:
s1, preparing single-cell suspension after stem cell pancreatin digestion;
s2, dripping the single-cell suspension liquid onto the surface of a collagen membrane, and placing the collagen membrane into an incubator for culture;
and S3, adding a culture medium for continuous culture to form a collagen membrane compound loaded with stem cells.
According to the disclosure, the collagen membrane is obtained by decellularizing pigskin, and has a double-layer structure; the length of the collagen film is 5-200mm, the width is 5-100mm, and the thickness of each layer of collagen film is 0.1-10mm.
According to the present disclosure, the surface of the collagen film bilayer structure includes a rough surface and a smooth surface, wherein the single cell suspension is dropped to the rough surface of the collagen film in step S2.
According to the present disclosure, in step S2, the seeding amount of the collagen membrane surface stem cells is 10 4 -10 7 Individual/cm 2 Preferably 4X 10 5 Individual/cm 2 。
According to the present disclosure, the stem cells are one or more selected from embryonic stem cells, multipotent induced stem cells, adult stem cells and mesenchymal stem cells;
the mesenchymal stem cells are one or more selected from bone marrow mesenchymal stem cells, adipose mesenchymal stem cells, umbilical cord mesenchymal stem cells and uterine blood mesenchymal stem cells.
According to the present disclosure, the culture conditions in the incubator may be conventional culture conditions during cell culture, preferably 37 ℃,5% co 2 The culture time is 1-7 days.
According to the present disclosure, the medium includes at least one of a stem cell medium, a differentiation medium, and a DEME medium.
Through the technical scheme, the stem cells are loaded on the surface of the collagen membrane to form the collagen membrane compound for preparing the cervical regeneration material. In the collagen membrane compound, the stem cells are used as active ingredients for treating cervical dysplasia, have strong self-renewal and differentiation potential, can be differentiated into cells of specific types of cervical tissues, promote regeneration, and can also improve the tissue microenvironment through paracrine action so as to promote tissue regeneration. The collagen membrane is used as a placement material after cervical angioplasty, has a double-layer structure, has good biocompatibility, and has a mechanical dilation and restenosis preventing effect on cervical tissues; meanwhile, the collagen membrane can be used as a stem cell supporting site, the cell activity, the dryness and the action time of the collagen membrane are maintained, and the collagen membrane can be degraded without secondary extraction. Thus, the present disclosure provides a treatment for cervical regeneration and further improves the therapeutic effect of cervical regeneration.
Additional features and advantages of the present disclosure will be set forth in the detailed description which follows.
Drawings
The accompanying drawings are included to provide a further understanding of the disclosure, and are incorporated in and constitute a part of this specification, illustrate the disclosure and together with the description serve to explain, but do not limit the disclosure. In the drawings:
fig. 1 is an electron microscopy image of a rough/smooth surface of a collagen film of the present disclosure.
Fig. 2 is a growth state of MSCs on collagen membrane in the collagen membrane composite prepared by the present disclosure (the massive structure in the figure represents living cells, no dead cells).
FIG. 3 is a comparative graph of the repair of full-thickness injury to the cervical of rabbits by the huMSCs collagen membrane complex (left: pre-treatment; right: post-treatment).
Detailed Description
Specific embodiments of the present disclosure are described in detail below with reference to the accompanying drawings. It should be understood that the detailed description and specific examples, while indicating and illustrating the disclosure, are not intended to limit the disclosure.
The present disclosure provides the use of a stem cell loaded collagen membrane complex prepared by a method comprising the steps of:
s1, preparing single-cell suspension after stem cell pancreatin digestion;
s2, dripping the single-cell suspension liquid onto the surface of a collagen membrane, and placing the collagen membrane into an incubator for culture;
and S3, adding a culture medium for continuous culture to form a collagen membrane compound loaded with stem cells.
Optionally, the collagen membrane is obtained by decellularizing pigskin, and has a double-layer structure; the length of the collagen film is 5-200mm, the width is 5-100mm, and the thickness of each layer is 0.1-10mm.
Alternatively, the surface of the collagen membrane bilayer structure comprises a roughened surface and a smooth surface, and the single cell suspension is dripped onto the roughened surface of the collagen membrane.
The collagen membrane provided by the disclosure has a double-layer structure, the internal structure and the thickness of each layer are suitable for tissue reconstruction, the growth requirement of natural tissue cells can be met, a supporting site is provided for tissue regeneration, and the cell activity, the dryness and the action time of the collagen membrane are maintained.
Optionally, in step S2, the seeding amount of the stem cells on the surface of the collagen membrane is 10 4 -10 7 Individual/cm 2 Preferably 4X 10 5 Individual/cm 2 。
The stem cells provided by the present disclosure are capable of secreting growth factors, providing active ingredients for tissue regeneration, and also improving local microenvironment through paracrine to promote tissue regeneration.
Optionally, the stem cells are one or more selected from embryonic stem cells, multipotent induced stem cells, adult stem cells and mesenchymal stem cells; preferably, the stem cells are mesenchymal stem cells;
the mesenchymal stem cells are one or more selected from bone marrow mesenchymal stem cells, adipose mesenchymal stem cells, umbilical cord mesenchymal stem cells and uterine blood mesenchymal stem cells.
Alternatively, in step S2, the single cell suspension is added dropwise to the surface of the collagen membrane, and the conditions for culturing in an incubator are conventional cell culture conditions, preferably 37 ℃,5% CO 2 The culture time is 1-7 days.
Optionally, the medium comprises at least one of a stem cell medium, a differentiation medium, and a DEME medium.
The present disclosure is further illustrated in detail by the following examples.
Example 1
Stem cell culture: huMSCs (human umbilical cord mesenchymal stem cells) isolation, purification, and culture steps: fresh umbilical cord was collected, surface blood was removed and small tissue pieces of about 0.5cm were cut using scissors; adding pancreatin, digesting for 30 minutes in an incubator, and observing the release degree of cells by a microscope; sucking out the digested supernatant, seeding the digested tissue blocks in a culture flask at intervals of about 1cm apart from each tissue block, and adding a stem cell culture medium for culturing for 2-3 days to enable the tissue blocks to be fully adhered; changing the culture medium every 2-3 days, and observing the growth of cells from tissue blocks; and taking out the tissue blocks for freezing and culturing the tissue blocks when the cells grow 70% of the culture bottle.
Cell planting process: the huMSCs and collagen membrane composite culture step: (collagen membrane is treated by decellularizing pig skin tissue to obtain a double-layer structure, as shown in figure 1, one layer of surface is smooth surface, the other layer of surface is rough surface, the length is 20mm, the thickness is 3mm, and the width is 10 mm) (1) culturing MSCs to 80% density (i.e. end of exponential growth phase); (2) Preparing cell suspension after pancreatin digestion, counting cells and regulating cell density to 2×10 6 individual/mL; (3) Immersing the collagen membrane in a DMEM culture medium overnight, and sucking the liquid in the collagen membrane to dryness by using sterile filter paper or sterile gauze the next day; (4) Uniformly dripping MSCs cell suspension onto the rough surface of soaked collagen membrane, and automatically adsorbing the cell suspension into collagen membrane per cm 2 Area of collagenDropwise adding 100 mu L of cell suspension into the membrane material; (5) Placing the collagen membrane loaded with the stem cells in a culture dish, and then placing the collagen membrane in a cell culture box for 4 hours until the cells adhere; (6) After cell adhesion, unadsorbed stem cells were gently washed off using PBS and added to complete medium, and after 5 days of culture, a collagen membrane complex loaded with stem cells was formed, wherein MSCs were grown on the collagen membrane matrix material in a state as shown in fig. 2 (the massive structure in fig. 2 indicates living cells, no dead cells were present).
Example 2
Carrying out a rabbit cervical full-layer injury repair experiment by using a collagen membrane compound loaded with MSCs:
(1) Control group: cutting off the whole cervical layer on the right side of the rabbit, and placing the rabbit into a urinary catheter; suturing two needles at the position deeper than the uterus by 2cm to fix the uterus and the urinary catheter, and cutting off the superfluous urinary catheter of the outwards extending part;
(2) MSCs composite group: the right cervical of the rabbit was excised and a ring of the collagen membrane complex loaded with stem cells was wrapped around the catheter end-to-end with suturing.
Transplanting the collagen membrane complex loaded with MSCs into a cervical dysplasia rabbit model:
(1) Anesthetizing rabbit according to 0.1mL/kg body weight, shaving, cutting skin, peritoneum and everting to expose bladder, and draining urine with syringe to expose vagina and uterus; (2) 2 x 1cm of collagen membrane composite loaded with MSCs, prepared as in example 1, was stitched into a column along the long diameter; (3) Cutting off the whole ectocervical level, and discarding the cut tissue; the sutured collagen membrane compound loaded with stem cells is contacted with the cervical excised in a flush way in a short diameter; the outer layer material and the cervical outer layer are sewed singly and intermittently; the inner layer material and the cervical inner layer are sewed singly and intermittently; turning the cervix uteri on both sides into vagina, and suturing the vaginal wall with 6-0 absorbable suture; 3-0 non-absorbable lines suture the peritoneal layer, the skin layer.
As shown in fig. 3, the collagen membrane composite loaded with MSCs stem cells can promote cervical regeneration, and the untreated side cervical in the left figure has no tissue regeneration; in the right panel, the cervical is regenerated to a height consistent with the contralateral normal cervix following treatment with a stem cell loaded collagen membrane complex.
The collagen membrane compound loaded with stem cells has the advantages of simple preparation method, low cost and capability of effectively promoting cervical regeneration.
The preferred embodiments of the present disclosure have been described in detail above with reference to the accompanying drawings, but the present disclosure is not limited to the specific details of the above embodiments, and various simple modifications may be made to the technical solutions of the present disclosure within the scope of the technical concept of the present disclosure, and all the simple modifications belong to the protection scope of the present disclosure.
In addition, the specific features described in the above embodiments may be combined in any suitable manner without contradiction. The various possible combinations are not described further in this disclosure in order to avoid unnecessary repetition.
Moreover, any combination between the various embodiments of the present disclosure is possible as long as it does not depart from the spirit of the present disclosure, which should also be construed as the disclosure of the present disclosure.
Claims (8)
1. Use of a stem cell loaded collagen membrane composite for the preparation of a material for cervical regeneration, wherein the stem cell loaded collagen membrane composite is prepared by a method comprising the steps of:
s1, preparing single-cell suspension after stem cell pancreatin digestion;
s2, dripping the single-cell suspension liquid onto the surface of a collagen membrane, and placing the collagen membrane into an incubator for culture;
and S3, adding a culture medium for continuous culture to form a collagen membrane compound loaded with stem cells.
2. The use according to claim 1, wherein the collagen membrane has a bilayer structure; the surface of the collagen film bilayer structure comprises a rough surface and a smooth surface.
3. The use according to claim 2, wherein in step S2 the single cell suspension is applied dropwise to the roughened surface of the collagen membrane.
4. Use according to claim 1, wherein the collagen film has a length of 5-200mm and a width of 5-100mm, and each layer of collagen film has a thickness of 0.1-10mm.
5. The use according to claim 1, wherein the collagen membrane is a collagen membrane obtained by decellularizing pigskin.
6. The use according to claim 1, wherein in step S2, the collagen membrane surface stem cells are seeded at an amount of 10 4 -10 7 Individual/cm 2 Preferably 4X 10 5 Individual/cm 2 。
7. The use according to claim 1, wherein the stem cells are one or more selected from embryonic stem cells, multipotent induced stem cells, adult stem cells and mesenchymal stem cells;
the mesenchymal stem cells are one or more selected from bone marrow mesenchymal stem cells, adipose mesenchymal stem cells, umbilical cord mesenchymal stem cells and uterine blood mesenchymal stem cells.
8. The use of claim 1, wherein the medium comprises at least one of a stem cell medium, a differentiation medium, and a DEME medium.
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| Application Number | Priority Date | Filing Date | Title |
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| CN202310301363.8A CN116459397A (en) | 2023-03-24 | 2023-03-24 | Application of collagen membrane compound loaded with stem cells in preparation of material for cervical regeneration |
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| CN202310301363.8A CN116459397A (en) | 2023-03-24 | 2023-03-24 | Application of collagen membrane compound loaded with stem cells in preparation of material for cervical regeneration |
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