Study On The Roles And Mechanism Of Mesenchymal Stem Cells Combined With PGS Scaffold Transplantation On Intrauterine Adhesion-injury Of Endometrium

Intrauterine adhesion(IUA)is a common acquired endometrial disease,which is mainly secondary to endometrial injury,including trauma,uterine curettage,infection,congenital abnormalities of the uterus,etc.In recent years,the incidence of IUA increased rapidly due to the increased endometrial injury,which has become one of the most important causes of infertility in women of reproductive age.Currently,the main therapeutic measure against IUA is transcervical resection of adhesion(TCRA)under hysteroscopy,and then to prevent the re-formation of adhesion using estrogen and Intra Uterine Device(IUD)after operation.However,for the severe IUA patients,the probability of endometrium re-adhesion after TCRA is still as high as 20%~62.5%,and the pregnancy rate is only 22.5%~33.3%.Thus,it is highly desired to develop alternative approaches to treat IUA.Stem cell therapy is a promising method for treating injury or disease.To date,scientists have explored the effects of different sources of stem cells on endometrial injury in preclinical animal models and clinical studies.In previous studies,stem cells were transplanted either by local in situ injection or by spiral arterial injection.However,the number of stem cells surviving in the site of endometrial injury after local in situ injection is limited,while the recruitment efficiency after spiral arterial injection transplantation is low,which lead to the low pregnancy rate after treatment.The researchers developed some kinds of methods to increase the survival rate of the transplanted stem cells.They found that some biomaterials such as PLGA scaffolds,hydrogels,and nano-structured lipid carriers can increase the survival rate of stem cells and therefore improve the effectiveness of stem cell therapy.Transplantation of collagen/BMSCs promotes the proliferation of endometrial and muscular cells,and recovers the capacity of endometrium to support the location and maturation of the embryos.However,the cell survival upon implantation is still limited.Furthermore,as one of the biogenic biomaterials,collagen demonstrated relatively poor reproducibility that has potential risks of delayed hypersensitivity,disease transmission,and immunogenic responses.Poly(glycerol sebacate)(PGS)is a synthetic bioelastomers and has been widely used in a variety of biomedical applications,especially for soft tissue regeneration.One of the most important features of PGS is its robust elasticity,which enables it to well sustain and restore various deformations of soft tissues in dynamic mechanical environments without mechanical irritations to the surroundings.Meanwhile,the beneficial effect of stem cells on restoration of endometrium after injury and its therapeutic mechanism remain unclear.In this study,we compares the effects of BMSCs-loaded elastic poly(glycerol sebacate)(PGS)scaffold with poly(lactic-co-glycolic acid)(PLGA)and collagen scaffolds in resumption of damaged rat uterus.Our results show that,the 3D architecture provided by PGS scaffolds favors the attachment and growth of rat BMSCs.In vivo bioluminescence imaging shows that compared with direct BMSCs intrauterine injection,PLGA,and collagen scaffolds,the PGS scaffold significantly prolongs the retention time of BMSCs in a wounded rat uterus model.It is found that the level of transforming growth factor?1(TGF-?1),basic fibroblast growth factor(bFGF),vascular endothelial growth factor,and insulin-like growth factors in the injured endometrium adjacent to PGS/BMSCs constructs is higher than those of rats receiving PLGA/BMSCs,collagen/BMSCs,or BMSCs intrauterine transplantation.Besides,transplantation of PGS/BMSCs leads to better morphology recovery of the damaged uterus than PLGA/BMSCs and collagen/BMSCs.The receptive fertility of PGS/BMSCs is 73.6±1.1%,significantly higher than 54.7±1.6%in collagen/BMSCs and 42.3±1.4%in PLGA/BMSCs.Taken together,PGS/BMSCs may be a promising candidate for preventing IUA.Currently,it is believed that stem cells probably restore the endometrium through reducing the local inflammatory damage by secreting anti-inflammatory cytokines and growth factors and inducing angiogenesis at the injured site of endometrium by the way of autocrine and paracrine.However,we do not understand the role of non-coding genes in the process that stem cells promote the recovery of endometrium after injury.Exosome is a kind of lipid double membrane structure containing specific proteins,lipids and nucleic acid,mediated the delivery of these signaling molecules among cells affecting the cell function which plays an extremely important role in many kinds of pathological and physiological process such as antigen presentation,tumor growth and migration,restoration of damaged tissue,etc.In this study,we found that BMSCs treatment of rats subjected to mechanical damage(MD)significantly increased microRNA 340(miR-340)levels in the regenerated endometrium.Then we employed knock-in and knock-down technologies to up-regulate or down-regulate the miR-340 level in BMSCs(miR-340~+BMSCs or miR-340~-BMSCs)and their corresponding exosomes,respectively to test whether exosomes from BMSCs mediate miR-340 transfer.We found that the exosomes released from the primitive BMSCs or miR-340~+BMSCs but not miR-340~-BMSCs increased the miR-340 levels in primary cultured endometrial stromal cells(ESCs)comparedwiththecontrol,respectively.Furtherverificationofthis exosome-mediated intercellular communication was performed using exosomal inhibitor,GW4869.Tagging exosomes with red fluorescent protein demonstrated that exosomes were released from BMSCs and transferred to adjacent ESCs.Compared with controls,rats receiving primitive BMSCs treatment significantly improved functional recovery and down-regulated collagen1?1,?-SMA and transforming growth factor(TGF)-?1.The outcomes were significantly enhanced by miR-340~+BMSCs treatment,and were significantly weakened by miR-340~-BMSCs treatment,compared with primitive BMSCs treatment.In vitro studies reveal that miR-340transferred from BMSCs suppresses the up-regulated expression of fibrotic genes in ESCs induced by TGF-?1.These data suggest that the effective antifibrotic function of BMSCs is able to transfer miR-340 to ESCs by exosomes,and that enhancing the transfer of BMSCs-derived miR-340 is an alternative modality in preventing intrauterine adhesion.