Preparation method and application of endometrial interstitial cell loaded PLGA-PEG-PLGA three-block temperature-sensitive hydrogel

Abstract

The invention relates to a preparation method of a PLGA (poly (lactic-co-glycolic acid))-PEG (polyethylene glycol)-PLGA (poly (lactic-co-glycolic acid)) three-block temperature-sensitive hydrogel. The preparation method comprises the following steps: carrying out steam sterilization on the PLGA-PEG-PLGA three-block temperature-sensitive hydrogel; sterile purified water is added into the PLGA-PEG-PLGA three-block temperature-sensitive hydrogel, and a hydrogel solution is obtained; transferring the hydrogel solution into a centrifugal tube through a pipette, and heating the centrifugal tube in an incubator; taking out the centrifugal tube, adding a sterile buffer solution when the hydrogel solution does not flow, and refrigerating; after an upper-layer sterile buffer solution is sucked out after a preset time interval, the upper-layer sterile buffer solution is poured into a precooled sterile buffer solution; sucking out the sterile buffer solution at the upper layer, and placing at the critical phase transition temperature of 2-23 DEG C, so that the hydrogel solution is changed from a solid state to a liquid state; a part of the liquid hydrogel solution is placed in an EP tube, and when the color is blue after the litmus solution is added, the hydrogel solution is alkaline. According to the technical scheme, on the basis that the physicochemical properties of the hydrogel are not changed, the hydrogel can be closer to the physiological state of a human body and is more suitable for cell survival.

Description

Technical Field

[0001] The present invention relates to the field of biomedical engineering technology, and in particular to a method for preparing a PLGA-PEG-PLGA triblock thermosensitive hydrogel loaded with endometrial stromal cells and application of the PLGA-PEG-PLGA triblock thermosensitive hydrogel loaded with endometrial stromal cells in preparing a drug for repairing damaged endometrium. Background Art

[0002] With the development of regenerative medicine, cell therapy has become a research hotspot for the treatment of various diseases. It injects cells isolated and cultured in vitro into the damaged part to repair it. However, injecting the cell suspension in situ will cause a large loss of the cell suspension, and less than 5% of the stem cells are implanted in the injection site. In order to improve the cell implantation rate, cell-combined hydrogels can prolong the retention time in the in situ injection site to a certain extent, thereby improving the success rate of cell transplantation.

[0003] Hydrogel is a high molecular polymer that can store a large amount of water and maintain a certain shape. It has a stable three-dimensional structure and can be used as a carrier for cells, growth factors and various drugs. It has been widely studied in the fields of ophthalmic drug delivery, local release of anti-tumor drugs, repair of orthopedic joints, and endometrial repair. Hydrogels are roughly divided into natural hydrogels and synthetic hydrogels. Natural hydrogels include collagen, chitosan, etc., which have poor mechanical capacity and unstable properties. Synthetic hydrogels can set different conditions according to needs to produce corresponding hydrogels, such as poly N-isopropylacrylamide, poloxamer and polyester. The ideal hydrogel for tissue repair should have the advantages of temperature sensitivity and degradability, so as to facilitate injection and avoid secondary surgery for removal.

[0004] At present, most of the hydrogels with the advantages of temperature sensitivity and degradability are acidic hydrogels, and when this type of hydrogel is used for cell culture, most of the cells cannot survive. The improved methods include acid-base neutralization, buffer solution dissolution and complex construction. Acid-base neutralization improves the pH of the hydrogel relatively quickly and changes the environment inside the hydrogel instantly, but more ions are introduced, which changes the osmotic pressure inside the hydrogel, which is not conducive to the survival of cells in the hydrogel. The buffer solution dissolution method did not significantly change the internal pH value. The complex construction method has the possibility of incomplete degradation, and it is still necessary to find suitable substances. Summary of the invention

[0005] The present invention aims to solve at least one of the technical problems existing in the prior art or related art.

[0006] To this end, the purpose of the present invention is to provide a preparation method and application of a PLGA-PEG-PLGA triblock thermosensitive hydrogel loaded with endometrial stromal cells, which can make the hydrogel closer to the physiological state of the human body and more suitable for cell survival without changing the physicochemical properties of the hydrogel.

[0007] To achieve the above object, the technical solution of the first aspect of the present invention provides a method for preparing a PLGA-PEG-PLGA triblock thermosensitive hydrogel loaded with endometrial stromal cells, comprising the following steps:

[0008] The PLGA-PEG-PLGA triblock thermosensitive hydrogel was steam sterilized;

[0009] Adding sterile purified water to the PLGA-PEG-PLGA triblock thermosensitive hydrogel to obtain a hydrogel solution;

[0010] Transferring the hydrogel solution into a centrifuge tube by a pipette and placing it in an incubator to heat;

[0011] Taking out the centrifuge tube, adding a sterile buffer solution when the hydrogel solution stops flowing, and then refrigerating;

[0012] After a preset time interval, the upper sterile buffer solution is sucked out and poured into the pre-cooled sterile buffer solution;

[0013] After the upper sterile buffer solution is sucked out, the solution is placed below the critical phase transition temperature (2-23° C.) to change the hydrogel solution from solid to liquid;

[0014] A portion of the liquid hydrogel solution is placed in an EP tube. When the color turns blue after adding litmus solution, it indicates that the hydrogel solution is alkaline.

[0015] In the above technical solution, preferably, the steam sterilization is high-pressure steam sterilization with a pressure of 103.4 kPa.

[0016] In any of the above technical solutions, preferably, the content of the hydrogel solution is 5%-20%.

[0017] In any of the above technical solutions, preferably, the hydrogel solution is transferred to a 10 ml-20 ml centrifuge tube by a pipette, and placed in a 30° C.-38° C. incubator for heating for 3 min to 5 min.

[0018] In any of the above technical solutions, preferably, the centrifuge tube is taken out and inverted for 20s-40s, and when the hydrogel solution stops flowing, 5-10 times the volume of sterile buffer solution is added and then refrigerated at 2°C-8°C.

[0019] In any of the above technical solutions, preferably, the sterile buffer solution includes HEPES buffer, PIPES buffer, MOPS buffer or phosphate buffer.

[0020] In the above technical solution, preferably, after 1 day, after the upper layer of the sterile buffer solution is sucked out for the first time, it is poured into the sterile buffer solution precooled at 2°C-8°C;

[0021] After 2 days, repeat the above steps again.

[0022] In the above technical solution, preferably, after 3 days, the upper layer of the sterile buffer solution is sucked out for the third time and then placed below the critical phase transition temperature to change the hydrogel solution from solid to liquid.

[0023] In the above technical solution, preferably, the preparation method stabilizes the pH range of the originally strongly acidic PLGA-PEG-PLGA triblock thermosensitive hydrogel to between 6.5 and 7, which is suitable for cell growth and proliferation without causing hydrogel disintegration.

[0024] The technical solution of the second aspect of the present invention provides an application of a PLGA-PEG-PLGA triblock thermosensitive hydrogel loaded with endometrial stromal cells (hESC) in repairing damaged endometrium. The PLGA-PEG-PLGA triblock thermosensitive hydrogel loaded with endometrial stromal cells is prepared by a preparation method provided by any technical solution in the first aspect of the present invention.

[0025] In the above technical scheme, preferably, the third generation endometrial stromal cells with good growth status are selected, and when the cell fusion degree reaches 90% under the microscope, digestion fluid is added to prepare a cell suspension with a density of 10,000-50,000 / μl; an appropriate amount of hydrogel improved by liquid exchange is absorbed, and the hydrogel with concentrations of 5%, 10%, 15%, and 20% is prepared using phosphate buffer; an appropriate amount of hydrogel is absorbed and fully mixed with the cell suspension; the final cell concentration is 1000-5000 / μl.

[0026] In the above technical solution, preferably, the repairing of damaged endometrium is to prolong the retention time of endometrial stromal cells in the uterus.

[0027] In any of the above technical solutions, preferably, the damaged endometrium is intrauterine adhesion or thin uterine cavity.

[0028] Compared with the prior art, the preparation method of the PLGA-PEG-PLGA triblock thermosensitive hydrogel loaded with endometrial stromal cells provided by the present invention and the application thereof have the advantages that: the high-pressure steam sterilization method can completely sterilize the hydrogel without changing the structure and phase change ability of the hydrogel, and the shortening of the dissolution and degradation time after sterilization is more conducive to the rapid preparation of the hydrogel and its degradation in the body; the liquid exchange method can effectively improve the internal pH value and osmotic pressure of the hydrogel without changing the original physical properties of the hydrogel, making it closer to the physiological state of the human body and more suitable for cell survival. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] The above and / or additional aspects and advantages of the present invention will become apparent and easily understood from the description of the embodiments in conjunction with the following drawings, in which:

[0030] Figure 1 The phase transition state diagram of 10% concentration PLGA-PEG-PLGA is shown;

[0031] Figure 2 The diagram shows the color change of litmus indicator and pH value determination in 10% concentration hydrogel before and after improvement;

[0032] Figure 3 The distribution of live and dead cells of hESCs in 10% concentration hydrogel before and after modification is shown;

[0033] Figure 4 The growth status and growth curve of hESCs in the improved hydrogel are shown;

[0034] Figure 5 Shown are stained smears of vaginal exfoliated cells in each estrus cycle of SD rats;

[0035] Figure 6 The diagram shows the gross morphological changes of the uterus at various time points after modeling in the control group and the experimental group;

[0036] Figure 7 The comparison of pathological morphological changes of rat endometrium at different time points after modeling is shown;

[0037] Figure 8 The statistical analysis charts of various indicators of rat endometrial pathological morphology at various time points after modeling are shown;

[0038] Fig. 9 The comparison of VEGF-A positive areas in rat endometrium at different time points after modeling is shown;

[0039] Fig.10 The results of the comparison of VEGF-A positive areas in the rat endometrium at various time points after modeling are shown.

[0040] Fig.11The in vitro imaging of DIR-labeled HESCs is shown, where:

[0041] A is the luminescence of PBS and HESCs in vitro under IVIS, and B is the fitting straight line;

[0042] Fig.12 Shown are in vivo imaging images of HESCs and HESCs-hydrogel cell scaffold complexes in IUA rats;

[0043] Fig.13 The comparison of the gross morphological results of the uterus of IUA rats at various time points after treatment and that of the uterus of the control group is shown;

[0044] Fig.14 The figure shows the pathological morphological changes of the endometrium of IUA rats at different time points after treatment and the control group, where:

[0045] A: Overall view of H&E staining; B: Local view of H&E staining; C: Overall view of Masson staining; D: Local view of Masson staining;

[0046] Fig.15 The figure shows the comparison of the repair indexes of damaged endometrium at different time points after IUA rat treatment, where:

[0047] A: Number of endometrial glands; B: Endometrial thickness; C: Percentage of endometrial fibrosis area;

[0048] Fig.16 The figure shows the comparison of VEGF-A positive areas in the endometrium of IUA rats at various time points after treatment with those in the control group, where: A: Overall view of immunohistochemical staining (50×); B: Local view of immunohistochemical staining (100×); C: Statistical analysis; Fig.17 Shown are the pregnancy status of IUA rats in each group after 24 days of treatment. DETAILED DESCRIPTION

[0049] In order to more clearly understand the above-mentioned purpose, features and advantages of the present invention, the present invention is further described in detail below in conjunction with the accompanying drawings and specific embodiments. It should be noted that the embodiments of the present application and the features in the embodiments can be combined with each other without conflict.

[0050] In the following description, many specific details are set forth to facilitate a full understanding of the present invention. However, the present invention may also be implemented in other ways different from those described herein. Therefore, the protection scope of the present invention is not limited to the specific embodiments disclosed below.

[0051] According to an embodiment of the present invention, a method for preparing a PLGA-PEG-PLGA triblock thermosensitive hydrogel loaded with endometrial stromal cells comprises the following steps:

[0052] The PLGA-PEG-PLGA triblock thermosensitive hydrogel was steam sterilized;

[0053] Adding sterile purified water to the PLGA-PEG-PLGA triblock thermosensitive hydrogel to obtain a hydrogel solution;

[0054] Transferring the hydrogel solution into a centrifuge tube by a pipette and placing it in an incubator to heat;

[0055] Taking out the centrifuge tube, adding a sterile buffer solution when the hydrogel solution stops flowing, and then refrigerating;

[0056] After a preset time interval, the upper sterile buffer solution is sucked out and poured into the pre-cooled sterile buffer solution;

[0057] After the upper sterile buffer solution is sucked out, the solution is placed below the critical phase transition temperature (2-23° C.) to change the hydrogel solution from solid to liquid;

[0058] A portion of the liquid hydrogel solution is placed in an EP tube. When the color turns blue after adding litmus solution, it indicates that the hydrogel solution is alkaline.

[0059] In the above embodiment, preferably, the steam sterilization is high-pressure steam sterilization with a pressure of 103.4 kPa.

[0060] In any of the above embodiments, preferably, the content of the hydrogel solution is 5%-20%.

[0061] In any of the above embodiments, preferably, the hydrogel solution is transferred to a 10 ml-20 ml centrifuge tube by a pipette, and placed in a 30° C.-38° C. incubator for heating for 3 min to 5 min.

[0062] In any of the above embodiments, preferably, the centrifuge tube is taken out and inverted for 20s-40s, and when the hydrogel solution stops flowing, 5-10 times the volume of sterile buffer solution is added and then refrigerated at 2°C-8°C.

[0063] In any of the above embodiments, preferably, the sterile buffer solution includes HEPES buffer, PIPES buffer, MOPS buffer or phosphate buffer.

[0064] In the above embodiment, preferably, after 1 day, after the upper layer of the sterile buffer solution is sucked out for the first time, it is poured into the sterile buffer solution precooled at 2°C-8°C;

[0065] After 2 days, repeat the above steps again.

[0066] In the above embodiment, preferably, after 3 days, the upper sterile buffer solution is sucked out for the third time and then placed below the critical phase transition temperature to change the hydrogel solution from solid to liquid.

[0067] In the above embodiments, preferably, the preparation method stabilizes the pH range of the originally strongly acidic PLGA-PEG-PLGA triblock thermosensitive hydrogel to between 6.5 and 7, which is suitable for cell growth and proliferation without causing hydrogel disintegration.

[0068] According to another embodiment of the present invention, a PLGA-PEG-PLGA triblock thermosensitive hydrogel loaded with endometrial stromal cells is used in repairing damaged endometrium. The PLGA-PEG-PLGA triblock thermosensitive hydrogel loaded with endometrial stromal cells is prepared by a preparation method provided by any technical scheme in the first aspect of the present invention.

[0069] In the above embodiment, preferably, the third generation endometrial stromal cells with good growth status are selected, and when the cell fusion degree reaches 90% under the microscope, digestion fluid is added to prepare a cell suspension with a density of 10,000-50,000 / μl; an appropriate amount of hydrogel improved by liquid exchange is absorbed, and the hydrogels with concentrations of 5%, 10%, 15%, and 20% are prepared using phosphate buffer; an appropriate amount of hydrogel is absorbed and fully mixed with the cell suspension; the final cell concentration is 1000-5000 / μl.

[0070] In the above embodiments, preferably, the repairing of damaged endometrium is to prolong the retention time of endometrial stromal cells in the uterus.

[0071] In any of the above embodiments, preferably, the damaged endometrium is intrauterine adhesion or thin uterine cavity.

[0072] Experimental data

[0073] 1.1 Construction and stability evaluation of the SD rat intrauterine adhesion model

[0074] 1.1.1 Experimental animals

[0075] Female SD rats weighing about 200 g and aged 8 to 10 weeks were selected. SPF grade, all experimental animals were provided by Henan Experimental Animal Center, and all were confirmed to have stable estrous cycles by continuous vaginal smears.

[0076] 1.1.2 Observation of the estrous cycle in rats

[0077] At 8-9 am every morning, moisten a cotton swab with physiological saline and insert it into the rat's vagina. After rotating clockwise for 2-3 weeks, take it out and smear it on a glass slide. Fix it with Dif staining reagent A solution, 20s→stain it with Dif staining reagent B solution, 10s→stain it with Dif staining reagent C solution, 20s→rinse it with slowly flowing water, and immediately observe the type, morphology and number of the rat's vaginal exfoliated cells under a microscope to determine whether the rat is in estrus.

[0078] 1.1.3 Construction of rat intrauterine adhesion model by mechanical infection double injury method

[0079] 1. Rats were randomly divided into two groups, namely the control group and the experimental group, according to the random number table. Vaginal secretions of rats were fixed and stained every morning and then observed under a microscope to determine whether the rats were in estrus.

[0080] 2. Select rats in estrus and use a respiratory anesthesia machine. Induce anesthesia with 2-3% isoflurane and maintain anesthesia with 1.5-2% isoflurane. Fix the rats in supine position on a small animal operating board. Prepare the skin in the middle of the lower abdomen for disinfection. Keep the rats breathing and heartbeat naturally during the operation.

[0081] 3. Make a midline incision in the abdomen, starting from the base of the thigh, with a length of about 2.5 cm. Open the abdominal wall layer by layer to fully expose both uteri.

[0082] 4. Experimental group: First, select one side of the uterus, cut an incision about 5 mm long at 1 / 3 of the distance from the bifurcation of the uterus, use a 2.5 mm diameter curette to scrape the uterine cavity until a more obvious roughness is felt and the surface of the uterus turns dark red. Then, place the cotton thread soaked in LPS solution for 24 hours in the uterine cavity of that side. Do the same with the contralateral uterus, return the uterus to the abdominal cavity, rinse the abdominal cavity with saline, suture the abdominal wall incision layer by layer, and pass one end of the LPS cotton thread through the abdominal incision and fix it on the skin. Remove it after 48 hours.

[0083] Control group: Only the abdominal cavity was opened, and the bilateral uterus was exposed and then put back into the abdominal cavity and the abdominal wall incision was sutured;

[0084] 5. After modeling, apply penicillin powder locally on the abdominal incision of the rat for 3 consecutive days to prevent wound infection

[0085] 6. On the 3rd, 7th, 14th and 21st days after modeling, 4 rats in the experimental group and 2 rats in the control group were killed respectively, and the gross morphology of the bilateral uterus was observed. They were immersed and fixed in 4% paraformaldehyde solution for subsequent H&E staining, Masson staining and immunohistochemistry for VEGF-A staining.

[0086] 1.1.4 H&E staining, Masson staining, and immunohistochemical staining of uterine tissue sections

[0087] 1.1.4.1 Preparation of paraffin sections

[0088] 1. Fixation: Use 4% paraformaldehyde to fix the treated rat uterus, take it out after 24 hours, and rinse it with 70% ethanol.

[0089] 2. Dehydration: The sections were placed in gradient concentrations of ethanol for dehydration: 70% ethanol, 1h → 80% ethanol 1h → 90% ethanol, 1h → 95% ethanol I, 30min → 95% ethanol II, 30min → 100% ethanol I, 30min → 100% ethanol II, 30min.

[0090] 3. Transparency: Place the slices in xylene, a mixture of equal amounts of anhydrous ethanol, xylene I, and xylene II for 20 minutes each for transparency treatment.

[0091] 4. Wax dipping: Place the transparent rat uterus in 60℃ paraffin for 1 hour.

[0092] 5. Embedding: Pour an appropriate amount of paraffin into the embedding box, and quickly place the uterine tissue with the cut surface facing down, straighten and flatten the tissue so that the tissue is filled with paraffin. After the paraffin cools and solidifies, take it out of the embedding box and dry it.

[0093] 6. Sectioning: Adjust the appearance of the wax block and perform continuous sectioning with a thickness of 5 μm. Use a glass slide to pick out the flattened sections in the 40°C water bath. Set the oven to 60°C and place the slides in the oven for 2 hours. To prevent detachment, place them in a 37°C constant temperature box overnight.

[0094] 1.1.4.2 H&E staining

[0095] 1. Dewaxing: Take an appropriate amount of xylene solution, put the slices in, and soak until completely dewaxed.

[0096] 2. Hydration: Place the dewaxed sections in anhydrous ethanol I, anhydrous ethanol II and 75% ethanol for 5 minutes each for hydration, and finally rinse with ddH2O.

[0097] 3. Staining: Hematoxylin staining for 10 min, rinse with ddH2O → differentiate with differentiation solution for 5 s, rinse with ddH2O → bluing with ammonia for 5 s, rinse with ddH2O → eosin staining for 10 min, rinse with ddH2O.

[0098] 4. Dehydration: Dehydrate the stained sections in 95% ethanol I, 95% ethanol II, 100% ethanol I and 100% ethanol II for 30 seconds each.

[0099] 5. Transparency: Place the slices in xylene I and xylene II in turn for 15 minutes each until they are fully transparent.

[0100] 6. Sealing: Use neutral gum to seal the slices.

[0101] 7. Observation: Place the slide under a microscope for observation.

[0102] 1.1.4.3 Masson staining

[0103] 1. Dewaxing of paraffin sections: Place the sections in environmentally friendly dewaxing solution I and environmentally friendly dewaxing solution II for 20 minutes each to remove wax. Then place them in anhydrous ethanol I, anhydrous ethanol II and 75% alcohol for 5 minutes each to gradually dehydrate, and finally rinse with distilled water.

[0104] 2. Staining: Immerse the sections in Masson A solution, soak overnight, rinse with running water → dye solution mixed with Masson B and C solutions in equal proportions, immerse for 1 minute, rinse with running water → differentiate with differentiation solution for a few seconds, rinse with running water → Masson D solution, immerse for 6 minutes, rinse with tap water → Masson E solution, immerse for 1 minute, do not rinse with water, drain slightly → Masson F solution, 2 to 30 seconds.

[0105] 3. Dehydration: 1% acetic acid rinse and differentiation → anhydrous ethanol dehydration.

[0106] 4. Transparency: put into the third cylinder of anhydrous ethanol, 5 minutes → xylene, 5 minutes to fully transparent.

[0107] 5. Sealing: Use neutral gum to seal the slices.

[0108] 6. Observation: Place the slide under a microscope for observation.

[0109] 1.1.4.4 Immunohistochemical staining

[0110] 1. Dewaxing and hydrating paraffin sections: Place the slides in an oven at 60°C to melt the paraffin, and place them in xylene I and xylene II for 15 minutes each to remove the wax. Subsequently, place the slides in anhydrous ethanol I, anhydrous ethanol II, 85% alcohol, and 75% alcohol for 5 minutes each to gradually dehydrate, and finally rinse with distilled water. After drying slightly, place them in a universal tissue fixative for fixation, and take them out and rinse them with water after 15 minutes.

[0111] 2. Antigen repair: Heat the pH value of 6.0 citric acid repair solution to boiling → put in the slices, heat for 3 minutes → exhaust the gas in the pot, cool → put the slices in PBS solution, shake and wash on a decolorizing shaker for 5 minutes, for a total of 3 times.

[0112] 3. Block endogenous peroxidase: Place the slices in 3% H2O2 solution, incubate for 25 minutes at room temperature in the dark, and wash with shaking as described above.

[0113] 4. Serum blocking: draw a circle around the tissue and add 10% goat serum on the tissue, incubate at 37°C for 30 minutes for blocking.

[0114] 5. Add primary antibody: shake off the blocking solution, add diluted HRP goat anti-rabbit VEGF-A antibody on the slice, and place in a wet box to incubate overnight at 4°C.

[0115] 6. Add secondary antibody: Shake and wash as above, spin dry and add secondary antibody to cover the tissue, incubate at room temperature for 50 minutes.

[0116] 7. DAB color development: Shake and wash as described above, spin dry and then add freshly prepared DAB color development solution into the circle until brown-yellow color is observed under the microscope, then rinse to stop color development.

[0117] 8. Re-staining of cell nuclei: Re-stain with hematoxylin for 3 minutes, wash with tap water → differentiate with differentiation solution for a few seconds, rinse with tap water → blue with bluing solution, rinse with running water.

[0118] 9. Dehydration and sealing: Place the slices in 75% alcohol, 85% alcohol, anhydrous ethanol I, anhydrous ethanol II, n-butanol and xylene I in turn for 5 minutes each to dehydrate and make them transparent. After drying, seal the slices.

[0119] 10. Microscopic examination: Place under a white light microscope to interpret the results.

[0120] 1.1.5 Modeling effect evaluation criteria

[0121] 1. Gross appearance of the uterus: Observe and compare the changes in the appearance of the uterus of rats in the experimental group and the control group at four different time points after mechanical infection and double injury.

[0122] 2. Endometrial morphology: The changes in the endometrial morphology of rats in the experimental group were observed and compared at four different time points after mechanical infection and double injury under H&E staining.

[0123] 3. Number of endometrial glands and thickness of endometrium: Under high-power microscopy, the number of glands on the H&E-stained sections of the experimental group and the control group at each time point was counted, and then the average value was calculated; under high-power microscopy, 4 different directions were randomly selected on each H&E-stained section to measure the thickness of the rat endometrial layer, and then the average value was calculated.

[0124] 4. Fibrosis area ratio: ImageJ image analysis software was used to analyze the endometrial fibrosis area ratio on Masson's staining sections of the experimental group and the control group at each time point (endometrial fibrosis area ratio = area of ​​stromal fibrosis / total area of ​​endometrial stroma and glands), and then the average value was calculated.

[0125] 5. Percentage of VEGF-A positive area: ImageJ image analysis software was used to analyze the percentage of VEGF-A positive area on the immunohistochemical staining sections of the experimental group and the control group at each time point, and then the average value was calculated.

[0126] 1.2 In vitro imaging of human endometrial stromal cells (hESCs)

[0127] 1. Select P3 hESCs. When the cells are observed to be 90% adherent under the microscope, discard the old culture medium, wash with PBS solution, add appropriate amount of trypsin, and when the cells become round and floating, add culture medium to terminate digestion. Centrifuge at 300g / min for 5min, discard the supernatant, add 1ml PBS solution, count, and adjust the concentration of cell suspension.

[0128] 2. Add an appropriate amount of prepared DIR working solution to the cell suspension and incubate in a 37°C, 5% CO2 incubator in the dark for 30 minutes.

[0129] Centrifuge at 300 g / min for 5 min, wash twice with PBS, and centrifuge the cells at 5 × 10 6 , 2×10 6 , 1×10 6 , 5×10 5 , 2×10 5 The cells were seeded in black 96-well plates, and PBS was used as a control well.

[0130] 4. Set the excitation light and receiving light intensity of the imager to Ex=745nm, Em=790nm, the exposure time to 10s, the fluorescence intensity to 60%, and immediately place the 96-well plate into the imager for observation.

[0131] 1.3 Tracking of hESCs and hESCs-hydrogel cell scaffold complexes in the uterus of IUA rats

[0132] 1. Stain P3 hESCs as described above and adjust the concentration of the cell suspension.

[0133] 2. Weigh a certain amount of the copolymer after high-pressure sterilization to prepare a 10% concentration hydrogel solution, and modify it according to the above-mentioned modification method for later use.

[0134] 3. SD female rats in estrus were selected, and the IUA rat model was established according to the mechanical infection double injury method mentioned above; one week after modeling, the rats were anesthetized under isoflurane induction, fixed on the operating board in the supine position, and the middle of the lower abdomen was prepared and disinfected. During the operation, the rats maintained natural breathing and heartbeat;

[0135] 4. hESCs group: Fully expose the bilateral uteri of two rats, clamp both ends of the uteri to prevent the cell suspension from flowing out of the uterine cavity, use a 40IU insulin needle to draw 100μl of cell suspension and inject it into one side of the uterine cavity along the long axis of the uterine cavity, be careful not to penetrate the uterine cavity, and do the same treatment on the opposite side. After a period of time, remove the clamps at both ends, slowly and gently rinse the abdominal cavity with saline, return the bilateral uteri to the abdominal cavity, and close the abdominal cavity layer by layer.

[0136] hESCs-PLGA-PEG-PLGA hydrogel cell scaffold complex group: fully expose the bilateral uteri of two rats, clamp the two ends of the uterus to prevent the scaffold from flowing out of the uterine cavity before gelation, and use a pipette to draw 100μl of the above-mentioned improved hydrogel and 10μl of cell suspension into the EP tube, mix them evenly, and use a 40IU insulin needle to inject into one side of the uterine cavity along the long axis of the uterine cavity. Also be careful not to penetrate the uterine cavity. Do the same treatment on the other side of the uterine cavity. Cover the surface of the uterus with gauze moistened with warm saline, set the temperature of the heating lamp to the critical gelation temperature of the hydrogel, heat the bilateral uterine cavities for an appropriate time, and remove the heating lamp, gauze and the clamping devices at both ends after observing that the hydrogel is in a solidified state. After flushing the abdominal cavity, close the abdominal cavity layer by layer;

[0137] 5. Imager settings As described above, put the anesthetized rats into the imager, start the program, and observe the imaging of the bilateral uteri of the cell group and the cell scaffold group. After the end, put the rats back into the cage, observe the rats' activities and foraging, and put them into the animal room environment after the situation stabilizes. After a certain time interval, the fluorescence imaging of the rats' body surface was detected again.

[0138] 1.4 Evaluation of the repair capacity of hESCs-hydrogel cell scaffold complex on the endometrium of IUA rats

[0139] 1.4.1 Experimental Grouping

[0140] 72 SD female rats weighing about 200g and aged 8 to 10 weeks were randomly divided into the following five groups, namely control group: sham operation group, experimental group: ① PBS group (PBS) ② hydrogel group (PLGA-PEG-PLGA) ③ cell group (hESCs) ④ cell scaffold group (hESCs-PLGA-PEG-PLGA), with a total of 4 time points, 2 rats in each time point in the control group and 4 rats in each time point in each experimental group. The rats in the control group received only the abdominal opening and closing operation, and the rats in the experimental group were given corresponding treatment 7 days after receiving the mechanical infection double injury treatment.

[0141] 1.4.2 Specific experimental plan for each group

[0142] 1. Every morning, vaginal secretions from each group of rats were smeared, stained, and observed to determine whether the rats were in estrus.

[0143] 2. Rats in estrus in each group were given the following different treatment measures

[0144] (1) Control group: only abdominal opening and closing operation was performed on the subjects without any treatment;

[0145] (2) Experimental groups: Rats in each group were treated with the mechanical infection double injury method described above for 7 days and then given different treatment measures:

[0146] 1) PBS group: 100 μl of PBS solution with pH = 7.4 was injected into each side of the uterine cavity;

[0147] 2) Hydrogel group: 100 μl of 10% liquid hydrogel was injected in situ into each uterine cavity, and the surface of the uterus was covered with gauze moistened with warm saline. The temperature of the heating lamp was set to the critical gelation temperature of the hydrogel, and the bilateral uterine cavities were heated for an appropriate time. After the hydrogel was observed to be in a solidified state, the heating lamp, gauze, etc. were removed;

[0148] 3) Cell group: 100 μl hESCs suspension was injected into each uterine cavity;

[0149] 4) Cell scaffold group: 100 μl of hESCs-PLGA-PEG-PLGA hydrogel cell scaffold complex was injected in situ into each side of the uterine cavity, and the rest of the operation was the same as the hydrogel group.

[0150] 3. After flushing the abdominal cavity with warm saline, return the bilateral uteri to the abdominal cavity and suture the abdominal wall layer by layer;

[0151] 4. On the 3rd, 8th, 18th and 24th days after treatment, 2 rats in the control group and 4 rats in the experimental group were killed, and the bilateral uterine specimens were taken and fixed in 4% paraformaldehyde for subsequent H&E staining, Masson staining and immunohistochemical staining to detect the expression of VEGF-A;

[0152] 5. The methods and steps of H&E staining, Masson staining and immunohistochemical staining are as described above.

[0153] Evaluation of hESCs-hydrogel cell scaffold complex on fertility restoration in IUA rats

[0154] Twenty SD female rats weighing about 200 g and aged 8 to 10 weeks were randomly divided into 5 groups according to the above grouping, with 4 rats in each group. The rats in each group were treated and given corresponding treatment according to the above method. 24 days after treatment, the rats in each group were caged with SD male rats at a ratio of 1:1 or 2:1. The first day of pregnancy was recorded when the female rats had vaginal plugs and sperm was observed on the vaginal secretion smear. On the 15th day after the formation of vaginal plugs, the rats were killed, the rat uterus was freed, and the number of pregnant uteri, the number of pregnant uteri gestational sacs, and the number of gestational sacs with a diameter of more than 1 cm were observed and counted.

[0155] 1.6 Statistical analysis

[0156] The results were expressed as mean ± standard deviation. SPSS25.0, GraphPadPrism8, ImageJ, FlowJo and other software were used for statistical analysis and drawing. The independent sample t test was used to compare the means of two independent samples, and the one-way ANOVA was used to compare the means among multiple groups. The LSD method was used for multiple comparisons between groups. P < 0.05 was considered statistically significant. (*: P < 0.05, **: P < 0.01, ***: P < 0.001).

[0157] Experimental Results

[0158] 1 Construction of hESCs and PLGA-PEG-PLGA hydrogel cell scaffold complex in vitro

[0159] 1.1 Sterilization of hydrogel and determination of gelation temperature

[0160] According to the experimental results of the previous experiment group, the yellow viscous PLGA-PEG-PLGA copolymer was sterilized by high-pressure steam, and an appropriate amount of sterile PBS solution was added to prepare a 10% concentration hydrogel. Because the critical gelation temperature of the copolymer was different in different batches, the critical gelation temperature of the hydrogel used in this experiment was determined to be 34°C by the small tube inversion method (such as Figure 1 ).

[0161] 1.2 Determination of pH value of hydrogel before and after improvement of liquid exchange method

[0162] The preliminary experiments of this experimental group confirmed that hESCs died in large numbers in unmodified hydrogels. By comparing the two improvement methods of liquid exchange and acid-base neutralization, the liquid exchange method was selected as the hydrogel improvement method. Figure 2 -As shown in A and B, after adding an appropriate amount of litmus indicator to the unmodified hydrogel, the color of the hydrogel changes from colorless to red. Combined with the color change range of the litmus indicator, it can be proved that the unmodified hydrogel is acidic and cells cannot survive in an acidic environment. After adding an appropriate amount of litmus indicator to the hydrogel improved by the liquid exchange method, the color changes from colorless to purple. Combined with the color change range of the litmus indicator, it can be proved that the improved hydrogel is weakly acidic, neutral or weakly alkaline, which is closer to the physiological pH of the human body and more in line with the living environment of cells. In order to obtain a more accurate pH value, a pH meter was used to measure the pH value of the hydrogel before and after the improvement, as shown below Figure 2As shown in Figure C, the pH value of the unmodified hydrogel was approximately 3.05±0.03, and the pH value after the liquid exchange method was improved was approximately 7.17±0.16. The pH value of the hydrogel after improvement was significantly improved compared with that before improvement, and the difference was statistically significant (P<0.001).

[0163] 1.3 Toxicity test of hydrogels to hESCs before and after improvement of liquid exchange method

[0164] hESCs were mixed with 10% hydrogel before and after the liquid exchange method was modified. The cells grown in the hydrogel for 2 days were stained using the Calcein-AM / PI double staining kit to perform a qualitative analysis of live and dead cells. The distribution of live and dead cells is shown in Figure 3 As shown. In the hydrogel before improvement, a large number of dead cells stained with PI red can be seen under a fluorescence microscope, while there are very few live cells stained with Calcein-AM green, almost no. On the contrary, in the hydrogel after improvement, a large number of live cells stained with Calcein-AM green can be seen, and there are very few dead cells stained with PI red, which indicates that the 10% hydrogel improved by the liquid exchange method provides a good living environment for cells (as shown in Figure 2). Figure 3 ).

[0165] 1.4 Detection of hESCs proliferation in hydrogels modified by liquid exchange method

[0166] hESCs were mixed with the modified 10% hydrogel, and the proliferation of hESCs in the hydrogel was observed under a microscope after culturing for 1, 2, 3, 4, 5, 6, and 7 days. Figure 4 As shown, as time goes by, the cells grow rapidly in the hydrogel and gradually reach a stable level. A large number of cell clusters can be seen under the microscope. At the same time, as the hydrogel continues to degrade, the cells gradually diffuse out of the hydrogel and grow by adhering to the wall, gradually covering the entire bottom of the culture dish.

[0167] 2IUA rat model construction and stability evaluation

[0168] 2.1 Changes in vaginal secretions during the estrous cycle of SD rats

[0169] like Figure 5 As shown in the figure, the characteristics of the cells in the vaginal secretions of SD rats in each period of the estrus cycle are as follows: in proestrus, nucleated epithelial cells are the main cells; in estrus, anucleated keratinocytes are the main cells; in metestrus, nucleated epithelial cells, anucleated keratinocytes, and leukocytes are present in equal numbers; in diestrus, leukocytes are the main cells. Each period appears regularly and continuously.

[0170] 2.2 Changes in gross morphology of rat uterus after modeling

[0171] The rats in the control group and experimental group in estrus were subjected to double injury treatments of abdominal opening only and mechanical infection, and the rats in each group were killed on the 3rd, 7th, 14th and 21st days of treatment to separate the uterus. The gross morphological changes of the uterus of the two groups of rats at different time points are as follows:

[0172] 1. Control group: The uterus of rats was "Y"-shaped at all time points, maintaining a normal shape, with basically regular thickness and smooth surface, and no obvious congestion or water accumulation.

[0173] 2. Experimental group: After 3 days, edema appeared in the distal end of the rat's uterus. After 7 days, edema could still be seen in the distal end of both uteri, and the area gradually increased, almost reaching 1 / 2 of the uterus on one side. After 14 days, edema appeared in the entire section of the uterus from the bifurcation on both sides. After 21 days, the rat's uterus showed obvious deformity, with or without edema in the distal end, and atrophy and thinning in the proximal end, completely losing its normal structure. (e.g. Figure 6 )

[0174] 2.3 Pathomorphological changes of rat endometrium after modeling

[0175] The specimens were stained with H&E and Masson staining, and the changes in the endometrium morphology of the two groups of rats at different time points were as follows:

[0176] 1. Control Group

[0177] (1) H&E staining showed that the uterine cavity was relatively regular and roughly oval in shape. The endometrial epithelial line was intact and smooth, and a single layer of high columnar epithelium covered the uterine cavity and glandular cavity. The glands were round or oval in shape, scattered in the endometrial stroma, and occasionally existed in clusters. Abundant microvessels were visible in the stroma. The endometrial epithelial layer, stroma, and muscle layer had clear boundaries, and the thickness of the endometrium was relatively uniform (e.g. Figure 7 -A).

[0178] (2) Masson staining shows that the endometrial epithelium, glands, and muscle layers are red, and the endometrial stromal collagen fibers are light blue and arranged in a relatively neat pattern. The boundaries of each layer can also be clearly seen on Masson staining (e.g. Figure 7 -B).

[0179] 2. Experimental Group

[0180] (1) H&E staining showed that the uterine cavity morphology after 3 days lost its regularity compared with the uterus in the control group. The integrity of the endometrial epithelial line was destroyed, part of the tall columnar epithelium disappeared, and was covered by a single layer of flat epithelium or low columnar epithelium, or the stroma was directly exposed on the surface of the uterine cavity; the number of glands in the stroma decreased; local microvascular dilation and congestion were seen, and inflammatory cell infiltration was also seen. As time went on, it was seen that the uterine cavity gradually lost its normal morphology, large pieces of scattered tissue fragments were seen in the uterine cavity, the tall columnar epithelium completely disappeared, and the stroma was exposed; the number of glands in the stroma decreased, and the remaining tall columnar epithelium of the glands disappeared, becoming vacuolated and losing its secretory function (such as Figure 7 -C).

[0181] 2.4 Evaluation of the extent of endometrial damage in rats after modeling

[0182] The number of glands and thickness of the endometrium of rats in the control group with only abdominal opening and the experimental group with mechanical infection and double injury at each time point were counted and analyzed under H&E staining. The counting results are shown in Tables 1 and 2. Compared with the rats in the control group, the number of glands and thickness of the endometrium in the experimental group at the four time points decreased significantly, and no recovery was observed with the extension of treatment time.

[0183] Table 1 Changes in the number of endometrial glands in IUA rats

[0184]

[0185] Table 2 Changes in endometrial thickness in IUA rats

[0186]

[0187] Table 3 Changes in the percentage of endometrial fibrosis area in IUA rats

[0188]

[0189] The statistical analysis results are as follows Figure 8 A, B, The number of glands in the endometrial stroma of the experimental group was significantly different from that of the control group at 3, 7 and 21 days after double injury treatment (P<0.05). The thickness of the endometrium was significantly different from that of the control group at the four time points after double injury treatment (P<0.05).

[0190] The collagen fiber deposition in each group was evaluated by Masson staining and the area of ​​collagen staining was statistically analyzed. The evaluation results are shown in Table 3. The percentage of fibrosis area in the experimental group was higher than that in the control group at the four time points. As the experimental time prolonged, no recovery was observed.

[0191] Statistical analysis results Figure 8C, The differences in the percentage of fibrosis area between the experimental group and the control group were statistically significant at 7, 14, and 21 days after treatment (P < 0.05). On the third day after treatment, the new collagen fibers may not have been fully formed, and the difference between the experimental group and the control group was not significant (P > 0.05).

[0192] 2.5 VEGF-A staining results of rat endometrium after modeling

[0193] Under immunohistochemical staining, VEGF-A is mainly expressed in the endometrial epithelium and glandular epithelial cells, and the positive product is brown-yellow granules. The expression of VEGF-A was detected by immunohistochemical staining of the uterine tissues of rats in each group. Fig. 9 As shown in Table 4, compared with the experimental group, the control group showed a more obvious VEGF-A positive area. The percentage of the positive area was determined using ImageJ software, and the results are shown in Table 4.

[0194] Table 4 Changes in the percentage of VEGF-A positive area in the endometrium of IUA rats

[0195]

[0196] The statistical analysis results are as follows Fig.10 As shown in the figure, there were differences between the experimental group and the control group after 3 and 7 days of treatment, and the differences were statistically significant (P < 0.05). As the injury time prolonged, the percentage of VEGF-A positive area in the endometrium of rats in the experimental group increased. After 14 and 21 days of treatment, the difference between the experimental group and the control group was no longer significant (P > 0.05).

[0197] Evaluation of the repair ability of 3HESCs-hydrogel cell scaffold complex on the endometrium of IUA rats

[0198] 3.1 Tracking of HESCs and cell-scaffold complexes in the uterine cavity of IUA rats

[0199] 3.1.1 Fluorescence labeling and in vitro imaging of HESCs

[0200] HESCs were labeled in vitro with DIR fluorescent dye and inoculated into corresponding wells according to the pre-set cell number gradient. Then, an imager was used for imaging. It can be seen that the expression of fluorescein was detected in the wells inoculated with HESCs, but not in the wells inoculated with PBS. The fluorescence intensity of each well was circled using Aura software. It can be seen that the fluorescence intensity increased with the increase in the number of cells. The regression line was fitted for the number of cells and the fluorescence intensity, and the regression line equation was obtained: Y = 162.8*X + 12857656, R2 = 0.986, indicating that the two have good linearity. The size of the fluorescence intensity on the surface of the IUA rat in the later stage can roughly reflect the number of cells remaining in the uterus (such as Fig.11 ).

[0201] 3.1.2 In vivo tracking of HESCs and HESCs-hydrogel cell scaffold complexes

[0202] HESCs and HESCs-PLGA-PEG-PLGA hydrogel cell scaffold complexes labeled with DIR dye were transplanted in situ into the uterus of rats treated with mechanical infection double injury for 7 days, and the two groups of rats were imaged in vivo using an imaging device. As time went on, it was observed that the fluorescence intensity of the body surface of both groups of rats gradually weakened, but the fluorescence intensity of the body surface of the rats in the HESCs group decreased faster. On the 5th day after transplantation, the fluorescence intensity was significantly weakened, and on the 18th day, the fluorescence expression had completely disappeared.

[0203] In contrast, the fluorescence intensity of the rats in the HESCs-PLGA-PEG-PLGA hydrogel cell scaffold complex group decreased significantly more slowly. On the 18th day after transplantation, fluorescence expression could still be observed on the body surface, and the fluorescence completely disappeared on the 24th day. This shows that the HESCs-PLGA-PEG-PLGA hydrogel cell scaffold complex can significantly prolong the retention time of HESCs in the uterine cavity (such as Fig.12 ).

[0204] 3.2 Repair effect of HESCs-hydrogel cell scaffold complex on the endometrium of IUA rats

[0205] 3.2.1 Gross morphological changes of IUA rats at different time points after treatment

[0206] The rats in the control group and experimental group in estrus were subjected to double injury treatment of laparotomy and mechanical infection. After 7 days, the uterus of the rats in the control group was not treated in any way. The rats in the experimental group were divided into groups for treatment as described in the method and received appropriate amounts of PBS solution, hydrogel solution, HESCs suspension, and HESCs-PLGA-PEG-PLGA hydrogel cell scaffold complex. On the 3rd, 8th, 18th, and 24th days after treatment, the rats in each group were killed and the uterus was isolated. The gross morphological changes of the uterus of the rats in the control group and the experimental group at each time point are shown in the figure below. Fig.13 shown.

[0207] 1. Control Group

[0208] Generally speaking, it can be seen that the uterus of the rats at the four time points still maintained a normal shape, showing an obvious "Y" shape, with the proximal and distal ends being basically the same in thickness, with a smooth surface and no congestion or edema.

[0209] 2. Experimental Group

[0210] (1) PBS group and hydrogel group

[0211] The changes in the gross morphology of the rat uterus in the PBS group and the hydrogel group at each time point were similar. After 3 days, edema was visible at the distal end of the uterus, and a normal uterine appearance was visible at the proximal end. As time went on, the degree of edema at the distal end gradually deepened, the area gradually increased, and obvious narrowing deformity gradually appeared at the proximal end.

[0212] (2) HESCs group and cell-scaffold complex group

[0213] The distal end of the uterus in the HESCs group showed varying degrees of edema at the four time points, but none exceeded the middle and outer third, and the proximal end of the uterus did not show obvious atrophy, deformity, or narrowing. The gross morphological changes of the uterus in the cell-scaffold complex group were similar to those in the HESCs group, but the area of ​​uterine edema was significantly smaller than that in the HESCs group, and after 24 days of treatment, the gross appearance of the uterus was similar to that of the control group.

[0214] 3.2.2Pathological changes of IUA rats at different time points after treatment

[0215] 1. H&E staining

[0216] (1) Control group: The uterine cavity was relatively regular in shape, roughly in the shape of an oblong. The endometrial tissue was convex, the endometrial line was continuous and complete, and the uterine cavity and glandular cavity epithelium were covered by a single layer of tall columnar cells. The glands were mainly distributed between the endometrial stroma, with a large number of them, and the shape was round or oval, and occasionally clustered. The stroma was rich in microvessels.

[0217] (2) Experimental group

[0218] 1) PBS group and hydrogel group

[0219] The pathological morphological changes of the uterus of the two groups of rats were similar. The endometrial line was no longer smooth and complete, and the exfoliated endometrial epithelium was seen in the uterine cavity, and the thickness of the endometrium decreased. Over time, the uterine cavity even showed atresia, and the high columnar epithelium of the endometrium was gradually completely replaced by a single layer of flat epithelium. The number of glands decreased, and the epithelium was gradually replaced by a single layer of flat epithelium, and glandular atresia was seen. The number of new microvessels was very small.

[0220] 2) HESCs group and cell-scaffold complex group

[0221] The two groups of rats had similar pathological morphological changes in the uterus. Three days after treatment, H&E staining showed that the uterine cavity was still significantly reduced, the epithelium was still a single-layer flat epithelium, the endometrial line was still not smooth and incomplete, and the number of glands did not increase significantly. On the 8th day, the size of the uterine cavity, the continuity and integrity of the endometrial line were restored, and new tall columnar epithelium appeared; the number of glands increased. On the 18th and 24th days, as the treatment time prolonged, the uterine cavity gradually increased, the endometrium gradually thickened, and the morphology gradually returned to normal. The endometrial line was smooth and complete, the epithelium gradually returned to a single-layer tall columnar epithelium, and the number of new microvessels increased; the number of glands in the stroma increased over time, and the epithelium also returned to tall columnar epithelium (such as Fig.14 -A, B).

[0222] 2.Masson staining

[0223] (1) Control group: The endometrial epithelium, glands, and myometrium appear red. The collagen fibers in the endometrial stroma appear light blue and are arranged in a regular pattern.

[0224] (2) Experimental group

[0225] 1) PBS group and hydrogel group

[0226] Masson staining revealed that the blue staining in the endometrial stroma deepened over time, indirectly reflecting that there were more blue collagen fibers deposited in the stroma, which were arranged in a disorderly and irregular manner.

[0227] 2) HESCs group and cell-scaffold complex group

[0228] Three days after treatment, the blue staining of the intimal stroma was still deep, and the arrangement of collagen fibers was still chaotic. On the 8th day, the blue staining was reduced, and the arrangement showed a certain regularity. On the 18th and 24th days after treatment, the degree of blue staining gradually decreased, which also reflected that the deposition of collagen fibers gradually decreased and the arrangement became more regular (such as Fig.14 -C, D).

[0229] 3.2.3 Evaluation of repair indicators related to damaged endometrium at different time points after IUA rat treatment

[0230] 3.2.3.1 Comparison of the number of endometrial glands and endometrial thickness at different time points after IUA treatment After H&E staining, the number of glands and endometrial thickness in the endometrium of rats in each group were statistically analyzed, as shown in Tables 5, 6 and Fig.15-A and B show that the number of endometrial glands and endometrial thickness of rats in each group at each time point were different, and the differences were statistically significant (all P < 0.05). Further LSD multiple comparisons between groups showed that the number of glands and endometrial thickness in the HESCs group and the cell scaffold complex group were higher than those in the PBS group and the hydrogel group at the four time points, and the differences were statistically significant (P < 0.05). As the treatment time progressed, on the 18th and 24th days after treatment, the number of glands and endometrial thickness in the cell scaffold complex group were close to those in the control group, and the difference was no longer statistically significant (P > 0.05), while the HESCs group had significant differences from the control group except that there was no significant difference between the HESCs group and the control group on the 18th day of treatment (P > 0.05). This shows that both the HESCs group and the cell scaffold complex group have a certain repair effect on the endometrium of IUA rats, but the repair ability of the cell scaffold complex is sustained and stable, which is better than that of HESCs alone.

[0231] Table 5 Changes in the number of endometrial glands in rats after IUA treatment

[0232]

[0233] Table 6 Changes in endometrial thickness in rats after IUA treatment

[0234]

[0235] 3.2.3.2 Comparison of the percentage of endometrial fibrosis area at different time points after IUA treatment in rats

[0236] ImageJ image analysis software was used to analyze the percentage of fibrosis area in the endometrium of each group of rats after Masson staining, as shown in Table 7 and Fig.15 -C shows that there were overall differences in the percentage of fibrosis area in the uterine membrane of each group at each time point, and the differences were statistically significant (P < 0.05). Further LSD multiple comparisons between groups showed that as the treatment time progressed, HESCs gradually diffused out of the hydrogel and the hydrogel gradually degraded, and HESCs gradually enriched in the rat uterus to play a repairing role. On the 8th, 18th and 24th days after treatment, the difference between the cell scaffold complex group and the control group narrowed and was no longer statistically significant (P > 0.05), and was significantly lower than the PBS group and the hydrogel group, with a significant difference (P < 0.05). Compared with the HESCs group, the percentage of fibrosis area in the cell scaffold complex group continued to decline steadily, and after 24 days of treatment, it was significantly lower than the HESCs group. This shows that the constructed cell scaffold complex has a sustained and stable improvement effect on endometrial fibrosis in IUA rats.

[0237] Table 7 Changes in the percentage of endometrial fibrosis area in rats after IUA treatment

[0238]

[0239] 3.2.4 Evaluation of VEGF-A expression at different time points after IUA treatment in rats

[0240] The isolated uterine tissues of rats in each group were subjected to immunohistochemical staining to detect the expression of VEGF-A. From the immunohistochemical staining diagram, it can be seen that, overall, compared with the four experimental groups, the control group showed more obvious brown-yellow positive areas, and in the comparison of the four experimental groups, the HESCs group and the cell scaffold complex group showed more obvious brown-yellow positive areas at the four time points compared with the PBS group and the hydrogel group (e.g. Fig.16 -A, B). ImageJ software was used to measure the percentage of VEGF-A positive areas in the five groups, and statistical analysis was performed. The results showed that the percentage of VEGF-A positive areas in each group at each time point was different overall, and these differences were statistically significant (P < 0.05). Further LSD multiple comparisons between groups showed that on the 8th, 18th and 24th days after treatment, the percentage of VEGF-A positive areas in the cell scaffold complex group was significantly higher than that in the PBS group and the hydrogel group, and the difference was statistically significant (P < 0.05). It is worth noting that the percentage of VEGF-A positive areas in the cell scaffold complex group continued to increase steadily, showing significant improvement after 24 days of treatment, and the difference between the control group and the control group was no longer statistically significant (P > 0.05). On the other hand, the percentage of VEGF-A positive areas in the HESCs group also showed significant improvement compared with the PBS group and the hydrogel group, but except for a slight increase after 18 days of treatment, it was lower than the cell scaffold complex group at the other time points. This result indicates that the constructed cell scaffold complex can repair the damaged endometrium of IUA rats by promoting the expression of VEGF-A, and the effect is better than that of HESCs alone (Table 8, Fig.16 -C).

[0241] Table 8 Changes in the percentage of VEGF-A positive area in the endometrium of IUA rats after treatment

[0242]

[0243]

[0244] 4.3 Evaluation of HESCs-hydrogel composites on fertility restoration in rats

[0245] The ultimate goal of treating intrauterine adhesions is to restore the patient's reproductive function. Therefore, in order to further verify the effect of in situ transplantation of HESCs-PLGA-PEG-PLGA hydrogel cell scaffold complex on the restoration of reproductive function in IUA rats, we co-housed the female rats in each group after 24 days of treatment with healthy male rats at a ratio of 2:1. After the 15th day of pregnancy, the uterus of each group of rats was removed to observe the pregnancy status, as follows Fig.17 As shown in Table 9. It can be seen that both sides of the uterus of the control rats were pregnant, the embryos were evenly arranged in the uterus, and most of the embryos were more than 1 cm in diameter; only one of the four rats in the PBS group, hydrogel group and HESCs group was pregnant in one side of the uterus, and the pregnancy rate was 12.5%, of which the number of embryos in the PBS group and the hydrogel group was 2, and the number of embryos exceeding 1 cm was 1-2. The uterus of the other three rats was obviously narrowed and thinned, and the normal uterine cavity morphology was lost; the number of embryos in the HESCs group was more than the other two groups, with 5 embryos, of which 2 developed more than 1 cm. In the cell scaffold group, 2 of the 4 rats were pregnant, and one of the rats had both sides of the uterus pregnant, with a pregnancy rate of 37.5%, 14 embryos, and 11 embryos that developed more than 1 cm. It can be seen that the pregnancy rate and the number of embryos larger than 1 cm in the cell scaffold group were significantly greater than those in the other three groups in the experimental group. This result indicates that in situ transplantation of HESCs-PLGA-PEG-PLGA hydrogel cell scaffold complex can partially restore the reproductive function of rats with endometrial injury.

[0246] Table 9 Number of pregnant uteri and embryos in rats after IUA treatment

[0247] Grouping Bracket set PBS group Hydrogel group HESCs group Cell Scaffold Group Number of uteri 8 8 8 8 8 Number of pregnant uteri 8 1 1 1 3 Pregnancy rate 100% 100％ 12.5％ 12.5％ 12.5％ 37.5％ Number of embryos 59 2 2 5 14 Number of embryos >1cm 56 2 1 2 11

[0248] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for preparing a PLGA-PEG-PLGA triblock thermosensitive hydrogel loaded with endometrial stromal cells, characterized in that: The steps include: The PLGA-PEG-PLGA triblock thermosensitive hydrogel was steam sterilized; Adding sterile purified water to the PLGA-PEG-PLGA triblock thermosensitive hydrogel to obtain a hydrogel solution; Transferring the hydrogel solution into a centrifuge tube by a pipette and placing it in an incubator to heat; Taking out the centrifuge tube, adding a sterile buffer solution when the hydrogel solution stops flowing, and then refrigerating; After a preset time interval, the upper sterile buffer solution is sucked out and poured into the pre-cooled sterile buffer solution; After the upper sterile buffer solution is sucked out, the solution is placed below the critical phase transition temperature of 2-23° C. to change the hydrogel solution from solid to liquid; A portion of the liquid hydrogel solution is placed in an EP tube. When the color turns blue after adding litmus solution, it indicates that the hydrogel solution is alkaline.

2. The preparation method according to claim 1, characterized in that: The steam sterilization is high-pressure steam sterilization with a pressure of 103.4 kPa.

3. The preparation method according to claim 1, characterized in that: The content of the hydrogel solution is 5%-20%.

4. The preparation method according to claim 1, characterized in that: The hydrogel solution was transferred into a 10 ml-20 ml centrifuge tube by a pipette, and placed in a 30° C.-38° C. incubator for heating for 3 min to 5 min.

5. The preparation method according to claim 1, characterized in that: The centrifuge tube was taken out and inverted for 20s-40s. When the hydrogel solution stopped flowing, 5-10 times the volume of sterile buffer solution was added and then refrigerated at 2°C-8°C.

6. The preparation method according to claim 1, characterized in that: The sterile buffer solution includes HEPES buffer, PIPES buffer, MOPS buffer or phosphate buffer.

7. The preparation method according to claim 5, characterized in that: After 1 day, the upper sterile buffer solution was aspirated for the first time and then poured into the sterile buffer solution precooled at 2°C-8°C; After 2 days, repeat the above steps again.

8. The preparation method according to claim 7, characterized in that: After 3 days, the upper sterile buffer solution was sucked out for the third time and then placed below the critical phase transition temperature to change the hydrogel solution from solid to liquid.

9. The preparation method according to claim 7, characterized in that: The preparation method stabilizes the pH range of the originally strongly acidic PLGA-PEG-PLGA triblock thermosensitive hydrogel to between 6.5 and 7, which is suitable for cell growth and proliferation without causing hydrogel disintegration.

10. Use of the PLGA-PEG-PLGA triblock thermosensitive hydrogel loaded with endometrial stromal cells prepared by the preparation method according to any one of claims 1 to 9 in repairing damaged endometrium.

11. The use according to claim 10, characterized in that: The third generation of endometrial stromal cells with good growth status were selected. When the cell fusion degree reached 90% under the microscope, digestive fluid was added to prepare a cell suspension with a density of 10,000-50,000 / μl; an appropriate amount of hydrogel improved by liquid exchange was absorbed and prepared into hydrogels with concentrations of 5%, 10%, 15%, and 20% using phosphate buffer; an appropriate amount of hydrogel was absorbed and fully mixed with the cell suspension; the final cell concentration was 1000-5000 / μl.

12. The use according to claim 10, characterized in that: The repairing of damaged endometrium is to prolong the retention time of endometrial stromal cells in the uterus.

13. The use according to any one of claims 10 to 12, characterized in that: The damaged endometrium is characterized by intrauterine adhesion and thin uterine cavity.

Images