| Research backgroundIntrauterine adhesions(IUA)are lesions of the endometrium,mainly caused by trauma or surgery,resulting in partial or complete uterine occlusion,accompanied by progressive uterine fibrosis,which can lead to infertility,repeated abortions,abdominal pain and other adverse pregnancy outcomesThe main purpose of IUA treatment is to reconstruct the uterine cavity and restore endometrial function,and Hysteroscopic transcervical resection of IUA was performed.Adhesion treatment is the standard method for the treatment of intrauterine adhesions,but the high recurrence rate and low pregnancy rate of postoperative adhesions are still the focus of attention in moderate and severe IUA patients.In recent years,the progress of Stem cell research in the field of biomedicine has provided new opportunities for the treatment of IUA.The treatment of intrauterine adhesions with mesenchymal Stem Cells(ADSCs)has become a hot spot in medical research.Adipose Derived Stem Cells(ADSCs)are abundant in source and can be isolated.It was obtained by in vitro culture and had strong differentiation ability.In addition,the adipose mesenchymal stem cells after repeated amplification function between strong,its low immunogenicity,research shows that it has anti-inflammatory and anti fibrosis effect,can inhibit the stem cell apoptosis and promote its regeneration,in the future can be used as a potential treatment of IUA,yet ectomesenchymal stem cell treatment for fat IUA curative effect and the mechanism is not fully clear the Pluronic F-127 is made of Polyoxyethylene(PEO)and Poly propylene oxygen is a non-ionic surface-active copolymer.It is a bioabsorbable and biocompatible scaffold material approved by FDA in the United States.It is reversible at temperature rise,and gradually dissolves in the body through simple surface absorption and removalLow antigenicity and biodegradability,related experiments show that when it is used in combination with intrauterine adhesion surgery,the anti-adhesion efficiency increases;However,for patients with moderate to severe IUA disease with severe basal layer damage and endometrial loss,endometrial regeneration remains a huge challenge due to the limited effectiveness of current intervention measures.So far,no studies have been found on the application of ADSCs and PF-127 hydrogel complex in endometrial regeneration and repair of moderate to severe IUA patients.In this study,by establishing a chemical injury IUA model similar to the pathological characteristics of clinical IUA,and by isolating and ex tracting ADSCs,the adipose-mesenchymal stem cell-PF-127 hydrogel complex was constructed to evaluate its influence on endometrial receptivity and evaluate its biological safety,and to explore its therapeutic effect in vivo and its possible repair mechanism.In order to provide a new idea for solving the problem of endometrial receptivity decline in moderate and severe IUA patients.Part I:Establishment and evaluation of intrauterine adhesion chemical injury model in New Zealand white rabbitsObjectiveTo construct a stable and effective animal IUA model which was consistent with the pathological characteristics of human IUA.Methods1.Sixty healthy female New Zealand white rabbits were selected and randomly divided into 4 groups:sham operation group with chemical injury group(10s)and sham operation group with chemical injury group(30s)and sham operation group with chemical injury group(60s),with 15 rabbits in each group.The remaining 20 female rabbits were randomly divided into sham operation group with chemical injury group(30s)and sham operation group with chemical injury group(30s),and the 60s group with chemical injury,5 rabbits in each group were subjected to reproductive function experimental research.After modeling for 7d,14d and 28d,rabbits in each group were sacrificed in batches,bilateral uteri were removed,and fixed by soaking in 4%paraformaldehyde for subsequent HE staining and Masson staining experiments.He staining and Masson staining were performed on the embedded sections of the uterine tissue.2.The gross morphological changes of the uterus were observed by light microscopy,and the number of endometrial glands,endometrial thickness and degree of fibrosis were detected and analyzed.3.Pregnancy experiment was conducted to detect the changes of pregnancy rate and increasing the number of embryo implantation in each model group,and then to evaluate the reproductive function of IUA rabbits with four model methods.4.Results1.The gross morphology of the rabbits in the sham operation group showed smooth and flexible uterine wall,uniform thickness,high elasticity,complete uterine structure,complete epithelial cell structure,and abundant mesenchymal glands.Compared with the sham operation group,on the 7th day after operation,the endometrial morphology of the 10s chemical injury group showed almost no significant changes,and the endometrial structure was basically complete.On the 14th day after operation,the uterus showed mild bleeding and interstitial edema.At 28 days after surgery,the collagen fibers of extracellular matrix were slightly increased,and the uterine cavity morphology and structure were relatively complete.In the 30s group,the uterine glandular cavity was dilated,capillaries became brittle and easily ruptured and bled,but the morphological damage of cells was not obvious.On the 14th day after operation,dense neutrophils could be seen in endometrial gland lumen,and the structure of endometrial epithelial and glandular epithelial cells was incomplete.In the 1min group,severe uterine edema,loose endometrial tissue structure,obvious endometrial necrosis and multiple microabscesses were observed 7 days after surgery.On the 14th and 28th days after surgery,there was no epithelial regeneration,neutropenia,and the luminal epithelial structure was completely destroyed,with varying degrees of degenerative necrosis and more serious exclaved interstitial fibrosis,and scattered microabscesses in both the mucous layer and the muscular layer.2.Detection of endometrial thickness undulate,normal endometrium surface uterus submucosal layer and base layer assumes the circular or elliptic three sets of damage model rabbit uterus uterus shape is irregular,expansion or contraction,missing some shallow muscularis,sparse glands,part of the injury has shed endometrial fragments.The endometrial thickness of each group was measured at 7,14 and 28 days after modeling.After 7 days and 14 days,the endometrial thickness of the 10s group and the 30s group decreased gradually(P<0.05);In the 30s group,the endometrial thickness increased at 28 days after modeling compared with that at 14 days(P<0.05),while in the 60s group,the endometrial thickness continued to decrease with significant difference(P<0.05).3.After HE staining,the serous membrane,muscle,endometrium and epithelium of the uterus in the control group were normal,and the tissue structure was continuous and complete.The epithelial cells are mostly single columnar,the endometrium is rich in tubular glands,and the interstitial cells are evenly distributed.The structure of endometrial stroma in the four groups was loose,with submucosal fibrosis,scattered columnar cells,few glands,and a large number of inflammatory cell infiltration in part of the stroma,accompanied by varying degrees of congestion and bleeding.The number of endometrial glands in each group was measured at 7,14 and 28 days after modeling.At 7 and 14 days after operation,the number of endometrial glands in the three groups decreased gradually compared with the sham operation group,with statistical significance(P<0.05).The number of endometrial glands in the group with 10s and 30s of chemical injury increased after 28 days compared with 14 days(P<0.05),while the number of endometrial glands in the group with 60s of chemical injury continued to decrease,with statistical significance(P<0.05).4.The epithelium,blood vessels,glands,and muscles of the normal uterus are stained red,while the endometrium and serosa are stained light blue,indicating low levels of collagen fibers.The dark blue staining area in the uterus of the four groups was enlarged,indicating hyperplasia of collagen fibers.Some endometrial fragments were scattered in the uterine cavity,the interstitial cells were disordered and the collagen fibers were unevenly distributed.Fibrosis area ratio of each group was measured at 7,14 and 28 days after modeling.After 7 days and 14 days,the area ratio of endometrial fibrosis in the three groups increased gradually,and the differences were significant(P<0.05).The area ratio of endometrial fibrosis in the groups of 10s and 30s after chemical injury was lower than that in the group of 14 d after 28 d(P<0.05),while the area ratio of endometrial fibrosis in the group of 60s after chemical injury continued to increase,the difference was significant(P<0.05).5.The changes of reproductive function of endometrium of female rabbits in sham operation group,chemical injury group of 10s,chemical injury group of 30s and chemical injury group of 60s were evaluated.According to the observation of uterine gross morphology and the results of HE and Masson staining test,we found that the endometrial injury of rabbits after 14 days of modeling was close to the pathological characteristics of moderate and severe IUA.For further verification,the female and male rabbits were mated in a cage after 14 days of modeling,and the pregnancy rate and the number of uterine embryo implantation were calculated.The results showed that compared with the sham operation group,the number of uterine embryo implantation and pregnancy rate of rabbits in the 30s and 60s chemically injured groups decreased significantly,with statistical significance(P<0.05);However,the number of embryo implantation and pregnancy rate in the chemically injured 10s group were not significantly decreased compared with the sham operation group,and the difference was not statistically significant(P>0.05).In addition,unlike the sham group,excessive chemical damage can impair the integrity of the endometrium and severely damage the fertile endometrium,leading to the failure of successful conception.ConclusionThe animal model of IUA established by chemical injury for 30s can result in the decrease of the number of endometrial glands,the increase of the rate of fibrosis,and the decrease of endometrial receptivity,which conforms to the clinical pathological indications of intrauterine adhesion,and can be used as an effective animal model of IUA.Part Ⅱ Isolation,culture and identification of ADSCsObjectiveTo isolate and culture rabbit adipose mesenchymal stem cells and study the biological characteristics of their growth and proliferation in vitro.Methods1.In aseptic conditions,subcutaneous adipose tissue from the groin of New Zealand white rabbits was taken,and ADSCs were isolated by collagenase I digestion method,then passed and purified and cultured.2.The multidirectional differentiation potential was verified by adipogenesis and osteogenesis induction,and the induced differentiation was identified by alizarin red and oil red O staining.3.CCK-8 cell proliferation experiment was used to draw the cell proliferation curve,and the cell viability of ADSCs under different concentrations and durations was detected to study its proliferation effect.Results1.The ADSCs were inoculated in the culture flask and began to attach 6h,and the adherent cells were removed at 24h.The isolated ADSCs showed typical fibroblast-like growth with uniform morphology,long findle-shaped,pseudopod,and swirling growth.2.Adipogenic osteogenesis induced differentiation:Under the effect of adipogenic inducer,translucent lipid droplets in the cytoplasm of ADSCs could be observed under 3d post light microscopy,and the number increased gradually with time 14 d at the end of the induction,most of the cytoplasm filled with lipid drops,oil red dyed into bright red cells of rabbits induced 21 d,lens cells was observed under the stratified growth and similar calcium nodules,round lens and alizarin red staining after 30d visible with obvious calcium deposits around.3.The proliferation curve of ADSCs reached a growth peak at 5-6 days,and the proliferation of the third generation of ADSCs was faster.ConclusionThe culture time of ADSCs in vitro is relatively long,and ADSCs can be passed many times,with good proliferation and multidirectional differentiation potential.In this experiment,we compared the ADSCs of the first,second,third and fourth generations,and found that the cells had the characteristics of exponential growth period,and the third generation cells had a higher proliferation rate,indicating that the ADSCs cultured in vitro had a strong proliferation ability and good stability.Part III:Efficacy evaluation of rabbit adipose-derived mesenchymal stem cells,PF-127 hydrogel and its combination therapy for IUAObjectiveTo investigate the dual repair effects of adipo-mesenchymal stem cells loaded with PF-127 hydrogel on endometrial injury and adhesion.Methods1.Dissolve the PF-127 hydrogel powder in the supplied solvent to 1%concentration.The gel solution was then placed in a flask,stirred in a water bath and cooled to 5℃ to prepare an injectable PF-127 hydrogel.The time required for gelation at 37℃ was measured in a constant temperature water bath.After gelation,the gelation was placed at 4℃ to identify its thermal reversibility.2.Ectomesenchymal stem cells fat load PF-127 water gel compound preparation PF127 water gel packages between adipose mesenchymal stem cells,extracted with 0.25%trypsin-EDTA adipose mesenchymal stem cells,the extraction and separation of cells with different concentrations of PF-127 mixes hydrogel,injected them into the womb by CCK8 cell proliferation in vitro experiments,plot of cell proliferation,ectomesenchymal stem cells to detect fat load PF-127 hydrogel complex biological activities and its proliferation effect.3.Cell proliferation curves were drawn by CCK-8 in vitro cell proliferation assay to detect the biological activity and proliferation effect of adipose mesenchymal stem cells loaded with PF-127 hydrogel complex.4.A total of 25 New Zealand white rabbits were divided into sham operation group,IUA model group,PF-127 hydrogel group,ADSCs and ADSCs hydrogel complex group,with 5 rabbits in each group.After 14 days of intervention,a hydrogel complex containing ASDCS was injected through the uterine horn.In this study,in order to reduce experimental errors,we set up a self-control by injecting PBS buffer into the injured uterus on the left side,while the injured uterus on the right side was treated with the above three intervention methods.In the sham operation group,laparotomy was only performed without any treatment.5.After 14 days of intervention treatment,rabbits in each group were sacrificed in batches,bilateral uteri were removed and fixed in 4%paraformaldehyde for subsequent HE staining and Masson staining experiments.6.He staining and Masson staining were performed on the embedded sections of the uterine tissue.The gross morphological changes of the uterus were observed by optical microscopy,and the number of endometrial glands,endometrial thickness and fibrosis degree were detected and analyzed.7.To evaluate the function of the damaged rabbit uterus in receiving fertilized eggs,providing adequate nutrition to the developing fetus and supporting the normal development of the embryo,the changes in pregnancy rate and increasing the number of embryo implantation in each model were detected by reproductive function test.The number and morphological changes of endometrium emitosis were observed by electron microscopy,and the effect of chemical injury on endometrium receptivity was detected.Results1.In the sham operation group,the uterine wall was smooth and flexible,the thickness was uniform,the elasticity was high,the uterine structure was complete,the epithelial cell structure was complete,and the interstitial glands were abundant.In the IUA model group,the uterus was irregular in shape and poor in elasticity,with obvious blood clots in the uterine cavity,dilated glandular cavity,brittle capillaries,easy to rupture and bleeding,and edema in the interstitium.In the ADSCs group,on the 7th day of treatment,dense neutrophils were observed in the endometrial gland lumen,the structure of the endometrial epithelial and glandular epithelial cells was incomplete,and mild uterine bleeding occurred.On the 14th day of treatment,there was almost no bleeding,but the endometrial end was uneven and the edge of cells was worn.On the 28th day of intervention treatment,some fibrous adhesions remained,and scattered microabscesses appeared in both mucosal and muscular layers.In the PF-127 hydrogel group,on the 7th day of treatment,the uterine cavity was irregular,dilated or atrophied,the glands were sparse,and some of the damaged sites had exfoliated intimal fragments,but the cell morphological damage was not obvious.On the 14th day after treatment,there was almost no intimal debris on the endometrial surface.On the 28th day after surgery,there was slight recovery,but there were still intrauterine adhesions.In the ADSCs-PF-127 hydrogel group,on the 7th day of treatment,mucus was secreted in the glandular cavity,cells were arranged neatly,and the intercellular connections were close.There was still mild interstitial edema,and the interstitial edema basically disappeared on the 14th day after treatment.The morphology of the harem cavity was basically restored at 28 days.2.Detection of the number of endometrium glands After HE staining,the serous membrane,muscle,endometrium and epithelium of the uterus in the sham operation group were normal,and the tissue structure was continuous and complete.The epithelial cells are mostly single columnar,the endometrium is rich in tubular glands,and the interstitial cells are evenly distributed.The structure of endometrial stroma in the four groups was loose,with submucosal fibrosis,scattered columnar cells,few glands,and a large number of inflammatory cell infiltration in part of the stroma,accompanied by varying degrees of congestion and bleeding.The number of endometrial glands in each group was measured on the 14th day of intervention.The results showed that the number of glands in the ADSCs-PF-127 hydrogel group was significantly higher than that in the PF-127 hydrogel group and the ADSCs group,and there was no significant difference in endometrial thickness between the PF-127 hydrogel group and the IUA model group before and after the intervention treatment.3.The endometrial fibrosis area ratio was measured The epithelial vascular glands and muscles of the uterus in the sham operation group were stained red,while the endometrium and serosa were stained light blue,indicating a low level of collagen fiber.The fibrosis area ratio of each group was measured on the 14th day of intervention treatmentOn the 14th day after intervention,the area of fibrosis in the combined treatment group was significantly higher than that in the PF-127 hydrogel group and the ADSCs cell treatment group,and there was no significant difference in the area of endometrial fibrosis between the PF-127 hydrogel group and the ADSCs cell treatment group compared with the IUA model group.4.The normal endometrial surface is wavy,and the submucosa and basal layer of the uterus are round or oval.Endometrial thickness was measured in each group on the 14th day of intervention.The results showed that the endometrial thickness in the combined treatment group was significantly higher than that in the PF-127 hydrogel group and the ADSCs cell treatment group,and basically returned to the normal level observed before the endometrial injury.There was no significant difference in endometrial thickness between the PF-127 hydrogel group and the ADSCs cell treatment group and the IUA model group.5.5.The pregnancy rate and number of pregnancy test evaluation To evaluate the changes in reproductive function of endometrium of female rabbits in sham operation group,IUA model group,PF-127 hydrogel group,ADSCs and ADSCs hydrogel complex group.After 14 days of intervention,female and male rabbits were mated in a cage,and the pregnancy rate and the number of uterine embryo implantation were calculated.The results showed that the number of uterine embryo implantation and pregnancy rate in the combined treatment group were significantly higher than those in the PF-127 hydrogel group and single cell treatment group(P>0.05),and there was no significant difference in endometrial thickness between the PF-127 hydrogel group and the single cell treatment group(P<0.05).ConclusionADSCS-PF-127 hydrogel complex can significantly improve endometrial pathological indexes,reduce fibrosis,promote endometrial growth,and improve endometrial receptivity,which can be used as an effective method for the treatment of IUA. |
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