Hydrogel Loaded Adipose-derived Mesenchymal Stem Cells Transplantation Repairs Intrauterine Adhesions In Rabbits

ObjectiveThe aim is to build a model of intrauterine adhesions(IUA)in New Zealand white rabbits.Rabbit adipose tissue-derived mesenchymal stem cells(rADSCs),pluronic F-127(PF)-127)Hydrogels and their composites,study and compare their repair effects on the endometrium,and explore feasible treatment strategies to promote the regeneration of damaged endometrium and improve pregnancy outcomes.MethodsPreparation and in vitro study of rADSCs-PF-127 hydrogel complex(Ⅰ)Isolation,cultivation and identification of rADSCs1.Aseptically collect fat from the groin of New Zealand white rabbits,isolate and extract rADSCs by collagenase digestion,adherent culture,and subculture and purify;2.Flow cytometry analysis to detect the immunophenotypes of rADSCs:CD90,CD34 and CD11b;3.Adipogenic and osteogenic induction culture of rADSCs to identify their multi-directional differentiation ability;4.CCK-8 method was used to detect rADSCs proliferation in vitro.(Ⅱ)Preparation of PF-127 Hydrogel and Study on Its Temperature Sensitive PropertiesA gradient concentration PF-127 hydrogel solution was prepared,and the time required for gelation at 37℃ was measured in a constant temperature water bath.After gelling,the gel was placed at 4 °C to identify its thermal reversibility.(Ⅲ)preparation of rADSCs-PF-127 hydrogel complexThe CCK-8 method was used to detect the biocompatibility of PF-127 hydrogel with rADSCs.The PF-127 hydrogel concentration with the best cell affinity was selected to encapsulate cells to prepare rADSCs-PF-127 hydrogel complex.Construction of New Zealand White Rabbit IUA Model and Evaluation of Modeling Effect(Ⅰ)Establishment of New Zealand White Rabbit IUA Model1.Adult female New Zealand white rabbits were randomly divided into a simple mechanical injury group and a mechanical infection double injury group,with 9 in each group;2.Two groups of New Zealand white rabbits were injured by mechanical curettage and mechanical curettage combined with foreign body infection to simulate the formation of IUA on one side of the uterus,and the other side of the uterus was used as a sham operation group for self-control without any treatment;3.New Zealand white rabbits were sacrificed in batches 7 days,14 days and 28 days after the modeling,and the two-horned uterus was collected.(Ⅱ)Evaluation of New Zealand White Rabbit IUA Modeling EffectThe endometrial morphology,the number of glands,and the proportion of fibrotic area were compared by HE staining and Masson staining,and the modeling method with better modeling stability was selected.rADSCs-PF-127 hydrogel composite transplantation repair IUA rabbit endometrium(Ⅰ)The mechanical damage infection double injury method was used to construct the New Zealand white rabbit IUA model,which was randomly divided into the following five groups:1.sham operation group,2.IUA model group,3.rADSCs group,4.Hydrogel group(PF-127),5.Complex group(rADSCs+PF-127),rADSCs,PF-127 hydrogel and rADSCs-PF-127 hydrogel complex were separately 7 days after the modeling Injected into the uterine cavity,14 days after transplantation,New Zealand white rabbits were sacrificed and the uterus was collected;(Ⅱ)Observe endometrial morphology and count glands by HE staining;(Ⅲ)Extraction of total RNA from uterine tissues and Real-time RT-PCR analysis to detect profibrotic signal pathway related factors(TGF-β1,Smad3),proinflammatory cytokines(IL-1β)and endometrial receptivity related factors(Integrin αvβ3,HOXA10)Expression at the gene level.Results1.rADSCs were successfully separated and extracted by collagenase digestion,and the cells were spindle-shaped after passage.Flow cytometry analysis showed that rADSCs positively expressed CD90(99.7%),negatively expressed CD34(1.72%)and CD11b(1.14%).By adding an induction differentiation medium,rADSCs can be induced to differentiate into adipocytes and osteocytes in vitro,which has multiple differentiation potentials.The third-generation rADSCs showed an "S" shape in vitro,and the growth rate was slower on the first day.It was an incubation period.The growth rate increased significantly on the 2-4th day and entered the logarithmic growth phase.The speed is flattening again,entering the plateau period,and the number of cells does not change much.2.Through simple mechanical curettage,the neointimal epithelium of New Zealand white rabbits basically achieved complete regeneration 7 days after injury,and 28 days after injury,New Zealand white rabbits’endometrium can basically achieve tissue reconstruction.The IUA model of New Zealand white rabbits established by the mechanical injury double injury method showed that the endometrial epithelium could not be completely regenerated 28 days after modeling.Compared with the IUA animal model constructed by mechanical injury alone,it is a This is a more stable and ideal model.3.The results of HE staining showed that compared with the sham operation group,the IUA model group had uterine cavity adhesions or even atresia,the smooth and intact cavity was damaged,and the number of glands was significantly reduced(P=0.000<0.05).However,compared with the uterus in the IUA model group,in the treatment group:the rADSCs group,the hydrogel group,and the complex group,the morphology of the damaged uterine cavity was improved,and the number of glands increased significantly(P<0.05).Compared with the rADSCs group(P=0.027<0.05)and the scaffold group(P=0.019<0.05),the number of glands in the New Zealand white rabbits in the complex group was larger.Real-time RT-PCR results showed that compared with the sham operation group,the expression levels of TGF-β1,Smad3 and IL-1βmRNA in the IUA model group increased significantly(P<0.05),while integrinαvβ3 3 and HOXA10 mRNA The expression levels of TGF-β1,Smad3 and IL-1β mRNA were significantly decreased in each treatment group(rADSCs group,hydrogel group and complex group)compared with the IUA model group(P<0.05).Significantly down-regulated(P<0.05),and the expression levels of integrin a vβ3 and HOXA10 mRNA significantly increased(P<0.05).ConclusionIntrauterine injection of rADSCs,PF-127 hydrogel,and their composites can promote the structural reconstruction and functional recovery of the damaged endometrium,and the combined effect is the best.