Experimental Study On Construction Of Tissue Engineering Endometrium By Thermosensitive Hydrogel

BackgroundIntrauterine adhesions(IUAs),also known as Asherman syndrome,can be accompanied by uterine scarring or thin endometrium.The clinical manifestations are non-specific,mainly thin menstruation,infertility and some pregnancy complications.At present,the clinical intervention measures for intrauterine adhesion mainly include transcervical resection of adhesion,postoperative balloon placement in the uterine cavity,and administration of anti-adhesion drugs and oral estrogen therapy.Traditional treatment methods have clinical limitations and cannot promote endometrial regeneration and improve fertility.In recent years,research has found that human endometrial mesenchymal stem cells(enMSCs)have a wide range of sources,low immunogenicity,no tumorigenicity,no ethical controversy,and autologous transplantation.A large number of studies at home and abroad have confirmed that transplantation of enMSCs by in situ intrauterine injection has a certain effect on repairing the endometrium,but the local colonization rate of mesenchymal stem cells is low,while in situ intrauterine transplantation of mesenchymal stem cells/scaffold complex(i.e.tissue engineering endometrium)contributes to the adhesion of mesenchymal stem cells to local injury sites,thus greatly improves the transplantation efficiency of mesenchymal stem cells.Poly-N-isopropylacrylamide hydrogel,as a new type of thermosensitive biological scaffold,has the advantages of good cell compatibility,can be loaded with a variety of biologically active factors,and can undergo sol-gel phase transformation with temperature,Its phase transition temperature is 32?.When the temperature is higher than 32?,it changes from liquid to gel,which facilitates cell colonization in the uterine cavity.When the temperature is lower than 32?,it changes from gel to liquid,which is convenient for cell injection,This process is reversible,which provides great convenience for cell culture in vitro and transplantation in vivo.There are few reports about the construction of tissue engineering endometrium with thermosensitive hydrogels at home and abroad.This study intends to construct tissue engineering endometrium using poly N-isopropylacrylamide thermosensitive hydrogel as a scaffold to improve the efficacy of autologous endometrial stem cell transplantation in the treatment of thin endometrium induced by intrauterine adhesion.ObjectiveTo construct a tissue engineering endometrium using human endometrial mesenchymal stem cells as seed cells and poly-N-isopropylacrylamide thermosensitive hydrogel as a scaffold to improve the success rate of autologous endometrial mesenchymal stem cells transplantation in the treatment of thin endometrium induced by intrauterine adhesion.Methods1.Tissue mass culture method was used to isolate enMSCs from the endometrium of patients with intrauterine adhesions and to culture and identify it in vitro.2.The enMSCs amplified in vitro were co-cultured with PNIPAM thermosensitive hydrogel and the cytotoxicity of the hydrogel was identified by flow cytometry and live-dead cell identification kit.3.enMSCs were co-cultured with PNIPAM thermosensitive hydrogel with different concentrations to find the optimal concentration suitable for in vitro culture of enMSCs.The tissue engineering endometrium constructed by thermosensitive hydrogel as scaffold was identified by CCK-8?live-dead cell identification kit and flow cytometry.Results1.enMSCs were successfully isolated and cultured from the endometrium of patients with intrauterine adhesions using tissue block culture.The primary cells reached 80%to 90%confluency in about 2 weeks.Cells are long spindle-shaped and grow in radial shape,and they could grow stably after passage.Flow cytometry results showed that enMSCs were positive for CD44,CD73,CD90,and CD 105,and negative for CD34,CD45,CD11b,CD19,and HLA-DR.2.The identification of live-dead cells showed that enMSCs had the best cell morphology in 2.5%PNIPAM thermosensitive hydrogel,which grew like cell clusters;flow cytometry showed that enMSCs had a cell survival rate of more than 95%on the 4th day of culture in thermosensitive hydrogel,and the hydrogel did not affect the expression of cell surface markers.3.CCK-8 results showed that the cells in the 2D culture group grew logarithmically in 1 to 4 days,and entered the plateau phase in 4 to 7 days.Cell proliferation in the 3D culture group was not significant within 7 days.On the 14th day of culture in PNIPAM thermosensitive hydrogel,the cells survived better than the 2D culture group.ConclusionPoly-N-isopropylacrylamide hydrogel has temperature sensitivity and good cell compatibility,and has low cytotoxicity to human endometrial mesenchymal stem cells.Tissue engineering endometrium constructed with this thermo-sensitive hydrogel as scaffold is beneficial to prolong the survival time of endometrial mesenchymal stem cells.