CN105983133A - Stem cell composite collagen scaffold kit used for repairing endometrial damage, and preparation method thereof - Google Patents
Stem cell composite collagen scaffold kit used for repairing endometrial damage, and preparation method thereof Download PDFInfo
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- CN105983133A CN105983133A CN201510051011.7A CN201510051011A CN105983133A CN 105983133 A CN105983133 A CN 105983133A CN 201510051011 A CN201510051011 A CN 201510051011A CN 105983133 A CN105983133 A CN 105983133A
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Abstract
The invention provides a stem cell composite collagen scaffold kit used for repairing endometrial damage, and a preparation method thereof. The stem cell composite collagen scaffold kit is composed of human umbilical cord mesenchymal stem cells and a collagen scaffold. The preparation method comprises the following steps: recovery of the umbilical cord mesenchymal stem cells, passage of the umbilical cord mesenchymal stem cells, and composite culture of the umbilical cord mesenchymal stem cells and the collagen scaffold. The kit provided by the invention employs collagen scaffold umbilical cord mesenchymal stem cells for endometrial repair and has the following advantages compared with traditional post-repair methods for endometrial damage: the collagen scaffold can provide attachment sites for the umbilical cord mesenchymal stem cells and allows the umbilical cord mesenchymal stem cells to be located on parts with endometrial damage, so repair of local tissue is benefited, repair effectiveness is improved and danger caused by cell migration is reduced; and the collagen scaffold can promote cell differentiation.
Description
Technical field
The invention belongs to obstetrics and gynecology field, a kind of stem cell for repairing endometrial impairment of specific design is multiple
Rubber alloy former scaffolding agent box and preparation method thereof.
Background technology
Inner membrance functional reparation obstacle, instead connective tissue proliferation reparation, uterus during endometrium major injury
Front and rear wall generation fibrosis, cicatrization, mainly show as intrauterine adhesion, amenorrhea and uterine not
Pregnant etc..How to rebuild the inner membrance of these patients and make it recover normal form and function is current regenerative medicine
An one of difficult problem.During research, scholars proposed multiple different therapeutic modality, as in utero saved
Educate device to use, foley's tube, the postoperative estrogen of adherence Separation or hyaluronic acid application etc., various treatment sides
Formula is respectively arranged with its pluses and minuses, makes a general survey of the result of document report, and these therapeutic effect exist disputes on and disappointing.
Therefore, novel method for the treatment of is needed badly for endometrial impairment.
Along with the discovery of human stem cell and the qualification of multiple separate sources, the treatment use probability of stem cell is also
Constantly it is being exploited, and is establishing this new branch of medicine of regenerative medicine, it is intended to utilizing stem-cell therapy mesh
Before still can not be by the disease of cured substance.
Mescenchymal stem cell (MSCs) be stroma source with mesenchymal stem cells MSCs (BM-MSCs)
Biological character is similar, has the cell of multi-lineage potential.MSCs is the adult stromal cell of pluripotency,
The tissue of multiple mesenchymal origin can be divided into, such as skeletonization, cartilage and adipose cell, in particular circumstances, go back
Can transdifferentiationof be that its hetero-organizations multiple such as myocyte, neurocyte, vascular endothelial cell, liver pancreatic cell are thin
Born of the same parents.Human umbilical cord mesenchymal stem cells (UC-MSCs) is i.e. the one of MSCs.Compared with BM-MSCs, UC-MSCs
SH2, CD106 and HLA-1 express low, OCT mrna expression is positive, and this explanation umbilical cord UC-MSCs is one
Plant more original MSCs group.Substantial amounts of experiment in vitro proves, under different inductive conditions, UC-MSCs is not only
Can to multiple mesoderma origin histiocyte break up, as osteoblast, chondrocyte, adipose cell,
Sarcoplast etc., it is also possible to divide to ectoderm and the neuron cell of endoderm origin and hepatocyte-like cells
Change.This points out us that UC-MSCs can be utilized to carry out the reparation of endometrium.
Due to the particularity of human uterus's cavity structure, the treatment of stem cell is utilized to be difficult to navigate to cell impaired
At inner membrance, easily there is tissue diffusion and loss, having of its clinical practice along with the flowing such as blood, body fluid in cell
Effect property and safety are restricted, and this requires to provide in local organization attachment for cell while repairing
Condition.Collagen is the main component of extracellular matrix, its have wide material sources, biocompatibility good, can
The advantages such as degraded, preparation technology are simple, experiment in vivo and vitro result all confirms that collagen scaffold can be that cell carries simultaneously
Supply attachment site and the differentiation of cell can be promoted.
Before making the present invention, there is no UC-MSCs and repair the correlational study report of endometrial impairment.Collagen scaffold
It is applied in clinic skin and oral mucosa reparation as a kind of biomaterial SFDA approval, has good
Biocompatibility, degradability and safety.
Summary of the invention
It is an object of the invention to the stem cell composite collagen scaffolding agent box for repairing endometrial impairment, for umbilicus
Band mescenchymal stem cell provides attachment site so that it is is positioned inner film injury position, beneficially local organization and repaiies
Multiple, improve and repair effectiveness and lower the danger that cell displacement brings.
A kind of stem cell composite collagen scaffolding agent box for repairing endometrial impairment, by human umbilical cord mesenchymal
Stem cell and collagen scaffold composition.
Described human umbilical cord mesenchymal stem cells is separated by heredity institute of Chinese Academy of Sciences stem cell bank and cultivates, through streaming
Cell art cell surface antigen identify: display CD14, CD34 and CD45 be feminine gender, CD29, CD44 and
CD105 is positive, is rendered as the feature of interstital stem cell.
Described collagen scaffold is provided by Zhenghai Biological Technology Co., Ltd., Yantai, and SFDA approval is applied to skin
Skin and oral mucosa reparation.
Described serious uterine inner film injury refers to the endometrium excessive damage that a variety of causes causes, inner membrance function
Property repair obstacle, instead connective tissue proliferation reparation, Uterus wall generation fibrosis, cicatrization.
It is a further object to provide a kind of above-mentioned stem cell for repairing endometrial impairment to be combined
The preparation method of collagen scaffold test kit, its technical scheme is as follows:
The preparation method of a kind of stem cell test kit for repairing endometrial impairment, comprises the following steps:
(1) human umbilical cord mesenchymal stem cells recovery: thawed by human umbilical cord mesenchymal stem cells, adds complete culture solution,
It is inoculated in culture bottle;
(2) human umbilical cord mesenchymal stem cells passes on: human umbilical cord mesenchymal stem cells above-mentioned steps obtained is cultivated,
Pass on;
(3) human umbilical cord mesenchymal stem cells and collagen scaffold compound criteria: collagen scaffold is cut out, takes previous step and obtain
The umbilical cord mesenchymal stem cells arrived, prepares single cell suspension after trypsinization, obtained cell suspension is uniformly added drop-wise to
On collagen scaffold, thus prepare the stem cell test kit for repairing endometrial impairment.
Comprising the concrete steps that of described step (1): take out the cryopreservation tube equipped with human umbilical cord mesenchymal stem cells, vertical
I.e. put in water-bath, shake so that it is thaw, cell suspension be transferred to centrifuge tube, add complete culture solution,
Mix homogeneously;Centrifugal, abandon supernatant;Add DMEM/F12 complete culture solution, be inoculated in culture bottle.
In described step (1), bath temperature is 40 DEG C, and centrifugal condition is: room temperature, 1200rpm, 5min.
Concretely comprising the following steps of described step (2): cultivate every two and half amounts and change liquid once, cultivates to attached cell
During 90% fusion, pass on;Absorb the old culture fluid in culture bottle;Add phosphate buffer (PBS),
It is shaken gently for culture bottle, to wash away the culture fluid remaining in bottle, absorbs phosphate buffer (PBS);Training
Add pancreatin in supporting bottle, be shaken gently for culture bottle, make Digestive system flow through all cells surface;Micro-being inverted
Microscopic observation, finds Cytoplasm retraction, after intercellular substance increases, adds complete culture solution immediately and terminates digestion;
Piping and druming bottle parietal cell repeatedly, forms cell suspension after cell detachment bottle wall;Cell suspension is sucked centrifuge tube,
Centrifugal, abandon supernatant;Adjust cell concentration with DMEM/F12 complete culture solution, be inoculated in culture bottle;It is placed in
Incubator is cultivated.
In described step (2), centrifugal condition is: room temperature 1200rpm, 5min.
In described step (2), it is placed in 37 DEG C, 5%CO2, saturated humidity CO2Incubator is cultivated.
In described step (2), adjusting cell concentration with DMEM/F12 complete culture solution is 2 × 105Individual cell/mL.
Concretely comprising the following steps of described step (3): collagen scaffold is cut into suitable size, UP faces down, and is placed in
In 6 orifice plates, being sufficiently humidified so as to by culture medium, inner air is removed in suitably extruding;With sterile gauze by unnecessary cultivation
Base blots;Take well-grown 5th generation umbilical cord mesenchymal stem cells, after trypsinization, prepare single cell suspension,
Count and adjust cell concentration;Obtained cell suspension is uniformly added drop-wise on collagen scaffold, and 6 orifice plates are put into cultivation
Case is cultivated.
In step (3), obtained cell suspension is uniformly added drop-wise on collagen scaffold, cell quantity about 1 × 106/cm2,
6 orifice plates are put into 37 DEG C, 5%CO2Incubator is cultivated 15min.
The invention has the beneficial effects as follows: in test kit of the present invention uses collagen scaffold umbilical cord mesenchymal stem cells to carry out
Film healing, has the advantage that compared with restorative procedure after tradition endometrial impairment collagen scaffold existence can
There is provided attachment site for umbilical cord mesenchymal stem cells so that it is be positioned inner film injury position, be conducive to local group
Knit reparation, improve and repair effectiveness and lower the danger that cell displacement brings;Collagen scaffold can promote simultaneously
Cell breaks up.Umbilical cord mesenchyma is dry to be carefully directly divided in internal film tissue or secretion cytokine profiles promotion
Film regeneration is repaired after all may participate in inner film injury, reaches to rebuild inner membrance purpose.Fill between institute of the present invention employment umbilical cord
Existing commercial prod, the collagen scaffold SFDA approval of matter stem cell is applied to skin and oral mucosa reparation
Technology is controlled, and safety is good, beneficially the scientific research of stem-cell therapy serious uterine inner film injury, protection
Laboratory Animal Welfare.
Accompanying drawing explanation
Fig. 1 is that flow cytometry umbilical cord mesenchymal stem cells surface markers measures;
Fig. 2 is postoperative 8,12 weeks substantially tissue appearance;
Fig. 3 is that in vitro tissue operative region HE dyeing in postoperative 8,12 weeks is observed;
Fig. 4 is postoperative 8,12 weeks in vitro tissue operative region smooth muscle Immunohistochemical studies;
Fig. 5 is postoperative 8,12 weeks in vitro tissue operative region body of gland Immunohistochemical studies;
Fig. 6 is postoperative 8,12 weeks in vitro tissue operative region blood vessel Immunohistochemical studies.
Detailed description of the invention
Below in conjunction with specific embodiment, the present invention will be further described.
Embodiment
For repairing the stem cell composite collagen scaffolding agent box of endometrial impairment, dry thin by human umbilical cord mesenchymal
Born of the same parents and collagen scaffold composition.
Human umbilical cord mesenchymal stem cells is separated by heredity institute of Chinese Academy of Sciences stem cell bank and cultivates, through fluidic cell
Art cell surface antigen is identified: display CD14, CD34 and CD45 are negative, CD29, CD44 and CD105
For the positive, it is rendered as the feature of interstital stem cell.
Collagen scaffold is provided by Zhenghai Biological Technology Co., Ltd., Yantai, SFDA approval be applied to skin and
Oral mucosa is repaired.
Serious uterine inner film injury refers to the endometrium excessive damage that a variety of causes causes, the functional reparation of inner membrance
Obstacle, instead connective tissue proliferation reparation, Uterus wall generation fibrosis, cicatrization.
The preparation method of the above-mentioned stem cell composite collagen scaffolding agent box for repairing endometrial impairment is:
Step 1, human umbilical cord mesenchymal stem cells is recovered: takes out cryopreservation tube, is immediately placed in 40 DEG C of water-baths, shakes
Dynamic, thaw rapidly;Cell suspension is transferred to 15mL centrifuge tube, adds 5mL complete culture solution, mixing;
Centrifugal (room temperature 1200rpm, 5min), abandons supernatant;Add DMEM/F12 complete culture solution, be inoculated in cultivation
In Ping.
Step 2, human umbilical cord mesenchymal stem cells passes on: cultivates every two and half amounts and changes liquid once, cultivates to adherent
When cell 90% merges, pass on;Absorb old culture fluid in culture bottle;Add PBS 1mL, shake gently
Dynamic culture bottle, to wash away the culture fluid remaining in bottle, absorbs PBS;Pancreatin 1mL is added, gently in culture bottle
Jog moves culture bottle, makes Digestive system flow through all cells surface;Observe under inverted microscope, find cell
Matter bounces back, and after intercellular substance increases, adds complete culture solution 1mL immediately and terminates digestion;Piping and druming bottle wall repeatedly
Cell, forms cell suspension after cell detachment bottle wall;Cell suspension is sucked centrifuge tube, centrifugal (room temperature
1200rpm, 5min), abandon supernatant;Adjusting cell concentration with DMEM/F12 complete culture solution is 2 × 105
Individual cell/mL, is inoculated in 25cm2In culture bottle, every bottle of 5mL;Put 37 DEG C, 5%CO2, saturated humidity
CO2Incubator is cultivated.
Step 3, human umbilical cord mesenchymal stem cells and collagen scaffold compound criteria: collagen scaffold is cut into suitable big
Little, UP faces down, and is placed in 6 orifice plates, is sufficiently humidified so as to by culture medium, and inner air is removed in suitably extruding,
With sterile gauze, unnecessary culture medium is blotted;Take well-grown 5th generation umbilical cord mesenchymal stem cells, pancreatin
Prepare single cell suspension after digestion, count and adjust cell concentration;Take appropriate cell suspension and be uniformly added drop-wise to glue
On former support, cell quantity about 1 × 106/cm2, 6 orifice plates are put into 37 DEG C, 5%CO2Incubator is cultivated
15min。
The test kit using the present invention to prepare is tested, and does nature reparation group, blank collagen scaffold reparation respectively
Group, umbilical cord mesenchymal stem cells collagen scaffold reparation group, carry out controlled trial, and result of the test is as follows:
Fig. 2-6 be respectively postoperative 8,12 weeks substantially tissue appearance, in vitro tissue operative region HE dyeing observe,
In vitro tissue operative region smooth muscle Immunohistochemical study, in vitro tissue operative region body of gland SABC
Dyeing is observed, in vitro tissue operative region blood vessel Immunohistochemical study.
In Fig. 2, postoperative 8 weeks of A, B, C;C, D, E 12 weeks after operation;Wherein, A, D
Natural reparation group;B, E blank collagen scaffold reparation group;C, F umbilical cord mesenchymal stem cells collagen
Support reparation group;
In Fig. 3, A-D: postoperative 8 weeks;E-H: 12 weeks after operation;Wherein, A, E normal uterine tissue;
B, F nature reparation group;C, G blank collagen scaffold reparation group;D, H umbilical cord mesenchymal stem cells
Collagen scaffold reparation group.A-H, 20 ×, length of the scale is 1000 μm.
In Fig. 4, A-D: postoperative 8 weeks;E-H: 12 weeks after operation;Wherein, A, E normal uterine tissue;
B, F nature reparation group;C, G blank collagen scaffold reparation group;D, H umbilical cord mesenchymal stem cells
Collagen scaffold reparation group.A-H, 20 ×, length of the scale is 1000 μm.
In Fig. 5, A-D, A '-D ': postoperative 8 weeks;E-H, E '-H ': 12 weeks after operation;Wherein, A,
A ', E, E ' normal uterine tissue;The natural reparation group of B, B ', F, F ';C、C’、G、
The blank collagen scaffold reparation group of G ';D, H umbilical cord mesenchymal stem cells collagen scaffold reparation group.A-H,
20 ×, length of the scale is 1000 μm, A '-H ', 100 ×, length of the scale is 500 μm.
In Fig. 6, A-D, A '-D ': postoperative 8 weeks;E-H, E '-H ': 12 weeks after operation;Wherein, A,
A ', E, E ' normal uterine tissue;The natural reparation group of B, B ', F, F ';C、C’、G、
The blank collagen scaffold reparation group of G ';D, H umbilical cord mesenchymal stem cells collagen scaffold reparation group.A-H,
20 ×, length of the scale is 1000 μm, A '-H ', 100 ×, length of the scale is 500 μm.
Claims (10)
1. the stem cell composite collagen scaffolding agent box being used for repairing endometrial impairment, it is characterised in that: by
Human umbilical cord mesenchymal stem cells and collagen scaffold composition.
2. the preparation method being used for repairing the stem cell test kit of endometrial impairment, it is characterised in that: include
Following steps:
(1) human umbilical cord mesenchymal stem cells recovery: thawed by human umbilical cord mesenchymal stem cells, adds complete culture solution,
It is inoculated in culture bottle;
(2) human umbilical cord mesenchymal stem cells passes on: human umbilical cord mesenchymal stem cells above-mentioned steps obtained is cultivated,
Pass on;
(3) human umbilical cord mesenchymal stem cells and collagen scaffold compound criteria: collagen scaffold is cut out, takes previous step and obtain
The umbilical cord mesenchymal stem cells arrived, prepares single cell suspension after trypsinization, obtained cell suspension is uniformly added drop-wise to
On collagen scaffold, thus prepare the stem cell test kit for repairing endometrial impairment.
3. the preparation method of the stem cell test kit for repairing endometrial impairment as claimed in claim 2, its
It is characterised by: comprising the concrete steps that of described step (1): take out frozen equipped with human umbilical cord mesenchymal stem cells
Pipe, is immediately placed in water-bath, shake so that it is thaw, and cell suspension is transferred to centrifuge tube, adds completely
Culture fluid, mix homogeneously;Centrifugal, abandon supernatant;Add DMEM/F12 complete culture solution, be inoculated in culture bottle
In.
4. the preparation method of the stem cell test kit for repairing endometrial impairment as claimed in claim 3, its
Being characterised by: in described step (1), bath temperature is 40 DEG C;Centrifugal condition is: room temperature, 1200rpm,
5min。
5. the preparation method of the stem cell test kit for repairing endometrial impairment as claimed in claim 2, its
It is characterised by: concretely comprising the following steps of described step (2): cultivate every two and half amounts and change liquid once, cultivates to patch
When parietal cell 90% is merged, pass on;Absorb the old culture fluid in culture bottle;Add phosphate buffer,
It is shaken gently for culture bottle, to wash away the culture fluid remaining in bottle, absorbs phosphate buffer;In culture bottle
Add pancreatin, be shaken gently for culture bottle, make Digestive system flow through all cells surface;See under inverted microscope
Examine, find Cytoplasm retraction, after intercellular substance increases, add complete culture solution immediately and terminate digestion;Repeatedly
Piping and druming bottle parietal cell, forms cell suspension after cell detachment bottle wall;Cell suspension is sucked centrifuge tube, centrifugal,
Abandon supernatant;Adjust cell concentration with DMEM/F12 complete culture solution, be inoculated in culture bottle;It is placed in incubator
Cultivate.
6. the preparation method of the stem cell test kit for repairing endometrial impairment as claimed in claim 5, its
Being characterised by: in described step (2), centrifugal condition is: room temperature, 1200rpm, 5min.
7. the preparation method of the stem cell test kit for repairing endometrial impairment as claimed in claim 5, its
It is characterised by: in described step (2), is placed in 37 DEG C, 5%CO2, saturated humidity CO2Incubator is cultivated.
8. the preparation method of the stem cell test kit for repairing endometrial impairment as claimed in claim 5, its
Being characterised by: in described step (2), adjusting cell concentration with DMEM/F12 complete culture solution is 2 × 105
Individual cell/mL.
9. the preparation method of the stem cell test kit for repairing endometrial impairment as claimed in claim 2, its
Being characterised by: concretely comprising the following steps of described step (3): collagen scaffold is cut into suitable size, UP faces down,
Being placed in 6 orifice plates, be sufficiently humidified so as to by culture medium, inner air is removed in suitably extruding;To be many with sterile gauze
Remaining culture medium blots;Take well-grown 5th generation umbilical cord mesenchymal stem cells, prepare slender after trypsinization
Born of the same parents' suspension, counts and adjusts cell concentration;Obtained cell suspension is uniformly added drop-wise on collagen scaffold, by 6 orifice plates
Put in incubator and cultivate.
10. the preparation method of the stem cell test kit for repairing endometrial impairment as claimed in claim 2,
It is characterized in that: in step (3), obtained cell suspension is uniformly added drop-wise on collagen scaffold, and cell quantity is about
1×106/cm2, 6 orifice plates are put into 37 DEG C, 5%CO2Incubator is cultivated 15min.
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CN106880871A (en) * | 2017-01-18 | 2017-06-23 | 烟台正海生物科技股份有限公司 | A kind of collagen leather material for promoting endometrium reparation and preparation method thereof |
CN107629998A (en) * | 2017-08-24 | 2018-01-26 | 浙江大学 | A kind of stem cell in vitro reparation with menstrual blood source is damaged the model of endometrium |
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CN112043727A (en) * | 2020-09-17 | 2020-12-08 | 山西宾大干细胞生物科技有限公司 | Preparation method and application of cell composite endometrium repair gel |
CN113134115A (en) * | 2021-05-08 | 2021-07-20 | 安徽医科大学 | Preparation method of collagen scaffold composite human menstrual blood stem cells for treating endometrial injury |
CN114470331A (en) * | 2021-12-31 | 2022-05-13 | 浙江金时代生物技术有限公司 | Preparation method of collagen scaffold composite umbilical cord stem cells for endometrial repair |
CN114832158A (en) * | 2022-05-10 | 2022-08-02 | 武汉理工大学 | Preparation method of composite mesenchymal stem cell-based skin injury repair scaffold material |
CN114949361A (en) * | 2021-12-31 | 2022-08-30 | 浙江金时代生物技术有限公司 | Preparation method of polylactic acid-glycolic acid copolymer scaffold for repairing endometrium and human umbilical cord mesenchymal stem cell compound |
CN115054678A (en) * | 2022-07-12 | 2022-09-16 | 康膝生物医疗(深圳)有限公司 | Preparation method and application of temperature-sensitive type collagen exosome composite hydrogel preparation for endometrial repair |
WO2024022079A1 (en) * | 2022-07-25 | 2024-02-01 | 京东方科技集团股份有限公司 | Use of human mesenchymal stem cell sheet in treatment of uterine scars |
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CN110408673A (en) * | 2019-07-01 | 2019-11-05 | 南通大学附属医院 | Study the method that Hsa_circ_0111659 repairs the mechanism of action in ESC in WJ-MSCs |
CN112043727A (en) * | 2020-09-17 | 2020-12-08 | 山西宾大干细胞生物科技有限公司 | Preparation method and application of cell composite endometrium repair gel |
CN113134115A (en) * | 2021-05-08 | 2021-07-20 | 安徽医科大学 | Preparation method of collagen scaffold composite human menstrual blood stem cells for treating endometrial injury |
CN114470331A (en) * | 2021-12-31 | 2022-05-13 | 浙江金时代生物技术有限公司 | Preparation method of collagen scaffold composite umbilical cord stem cells for endometrial repair |
CN114949361A (en) * | 2021-12-31 | 2022-08-30 | 浙江金时代生物技术有限公司 | Preparation method of polylactic acid-glycolic acid copolymer scaffold for repairing endometrium and human umbilical cord mesenchymal stem cell compound |
CN114470331B (en) * | 2021-12-31 | 2022-12-02 | 浙江金时代生物技术有限公司 | Preparation method of collagen scaffold composite umbilical cord stem cells for endometrial repair |
CN114832158A (en) * | 2022-05-10 | 2022-08-02 | 武汉理工大学 | Preparation method of composite mesenchymal stem cell-based skin injury repair scaffold material |
CN115054678A (en) * | 2022-07-12 | 2022-09-16 | 康膝生物医疗(深圳)有限公司 | Preparation method and application of temperature-sensitive type collagen exosome composite hydrogel preparation for endometrial repair |
WO2024022079A1 (en) * | 2022-07-25 | 2024-02-01 | 京东方科技集团股份有限公司 | Use of human mesenchymal stem cell sheet in treatment of uterine scars |
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