Effect Of Thermosensitive Hydrogel Containing Stromal Cell-derived Factor-1α On Intrauterine Adhesion And Endometrium Regeneration

Background and Purpose:Intrauterine adhesion(IUA)is a disease secondary with mechanic and inflectional injury,it will lead to IUA formation when the normal repair mechanisms turn to be abnormal.Current therapies are hysteroscopic adhesiolysis conbinating with the administration of hormonal drugs,but the patients of severe adhesion often suffer re-adhesion due to the lack of an effective barrier in uterine and endometrial basal layer damage.After surgery,the decomposition of intrauterine scaffold can partially prevent from the re-adhesion,but there is a limited effect and it’s easy to cause retrograde infection.In addition,it is more serious that the patients of severe adhesion often suffer re-adhesion due to endometrial basal layer damage.Therefore,it is urgent to research for more safe and effective method which is excellent both in barrier role and the induction of stem cell homing for repairing endometrial basal layer damage.The choice of scaffold material is the foundation of the intrauterine slow-release scaffold research.Biodegradable polymer materials is one of the hot research topic in the field of tissue engineering,and the thermosensitive hydrogel can make intelligent response to the ambient temperature to make transformation between sol phase(liquid)and gel phase.It can maintain liquid in vitro under low temperature environment,and transform to semi-solid after injection into uterinecavity through cervix,thus ideal barrier effect.What’s more,bioactive factors can be loaded to form a drug delivery system,so it has got a wide attention in the field of tissue engineering applications.In recent years,some researchers transplanted Bone Marrow Mesenchymal Stem Cells(BM-MSCs)as seed cells to endometrial lesions in mice,and found that the BM-MSC can promote the repair of endometrial injury,however,the separation,purification and amplification of MSCs is difficult in the clinical application.It can be avoid by "Stem cell homing" method,which refers to stem/progenitor cells in the circulation migrate to the target tissue or bone marrow."Homing" describes the characteristics of stem/progenitor cell migration and is a multi-step coordinated process,involving many cell factors,chemical factors and extracellular matrix,and it’s mainly through a series of stem cell homing axis.CXCL12-CXCR4 axis is widest researched at present.CXCL12(SDF-1)is sensitive to hypoxia,and stem/progenitor cells home to tissue according to SDF-1 concentration to promote tissue regeneration.So we assume that:We can inject thermosensitive hydrogel into uterine as a barrier,and load the recombinant human stromal cells-derived factor 1α(rhSDF-1α)among them as chemotactic factor to lead BM-MSCs homing and promote endometrial regeneration.We believe it’s may be postoperatively ideal solutionof severe IUA.1.Experimental method1.1 Protein control release experiment of PLEL-BSA composite hydrogelWe used solid PDLLA-PEG-PDLLA(PLEL)three block polymers to make up of 20 wt% hydrogel whichwas heated up to 37℃to test its transformation ability from gel phase to the sol phase.PLEL hydrogel was physically mixed withbovine serum albumin(BSA)to make PLEL-BSA composite hydrogel,then put it in37℃incubator,period of time to collect the release of BSA,and determinedtherelease rate of calculated BSA.1.2 Chemotaxis experiment of rhSDF-1α to MSCsWe cultured and identified BM-MSCs of New Zealand rabbits,then verified the chemotaxis of rh SDF-1α to BM-MSCs using transwell system.PLEL-rh SDF-1α composite hydrogels were prepared for animal experiment: 11 female adult New Zealand rabbits(22 uterus)were divided into 4 groups: spontaneous regeneration group(n = 6)which was treated by cutting the uterus lengthwise,removing the endometrium with scissors,and then suturing the incision after hemostasis,PLEL treatment group(n = 6)which was treated by injecting PLEL hydrogel after suturing the incision,PLEL-rh SDF-1α treatment group(n=6),which was treated by injecting PLEL-rhSDF-1α hydrogel after suturing the incision,and normal control group(n = 4)which just exposed the uterus via an abdominal midline incision.MSCs homing effect were estimated between PLEL-rhSDF-1α treatment group and PLEL treatment group after 7 days.1.3 Prevention effect on intrauterine adhesions and promotionon endometrial regeneration of PLEL-rh SDF-1αThe same method was applied in animal experiment.HE and immunohistochemical staining were made after 28 days.2.Results2.1 Phase transition characteristics of PLEL hydrogel and cumulative release curve of PLEL-BSA composite hydrogelPLEL hydrogel transform from gel to sol state when the temperature rise to(32-35℃).Our in vitro experiment verified that PLEL-BSA composite hydrogel posited in PBS at 37℃ can keep its shape more than 3 days.Cumulative release curve of PLEL-BSA composite hydrogel shows that BSA was releasedslowlymore than 3 days.2.2 Chemotaxis of rhSDF-1α to BM-MSCsThe migration of MSCs in vitro experiments show that the experimental group was 54.330 ±9.424,and the control group were 26.070±7.285 and 27.730±6.649,7.285 mm visible transwell little room under the room only to join rh SDF-1α,BM-MSCs migration quantity increased with a statistically significant difference(P < 0.05)compared with the control group for the presentation of rh SDF-1α.After 7 days,the immunofluorescence staining of CD29 and CD44 showed that there is MSCs homing to the regenerative endometrial tissue,however the quantity of PLEL-rh SDF-1α treatment group is significantly lager than PLEL treatment group.2.3 Effect of PLEL-rh SDF-1α hydrogelto intrauterine adhesions preventing and endometrial regeneration promotingAfter 28 days,epithelial tissue was not regenerated nearly in the spontaneous regeneration group,but two treatment groups epithelial layers and uterine cavities.ConclusionThermosensitive PLEL hydrogel containing rh SDF-1αis effective on preventing intrauterine adhesion and promoting the regeneration of endometrium.