A Novel 3D Bio-printed Cell-laden Bi-layer Scaffolds For Rat Uterine Regeneration

Serious injuries,followed by dilation and curettage(D & C)once damage the basal layer of uterus,often lead to intrauterine fibrosis,uterine scar formation,adhesion and so on,which may result in infertility in reproductive age women.Nowadays,the treatments,including preventing fibrosis,adhesion and promoting angiogenesis in the damaged area,are only suitable for small area injury and thin endometrium,and there is no effective method to repair large area and full-thickness uterus injury.Tissue engineering has emerged as one of the promising approaches for uterine regeneration.Proper biomimetic-engineered scaffolds are crucial in allowing functional regeneration of uterus,which contain multilayer complex endometrium,myometrium,and serosa.This study demonstrated a 3D bio-printed,cell-laden,bi-layer scaffold for functional uterine regeneration.3D bio-printed endometrial stromal layer(functional layer)and monolayer epithelium were composited to build bi-layer biomimetic scaffolds.We used two natural polymers,sodium alginate and hyaluronic acid,to form the composite bio-ink for printing.This new-type scaffold showed superior elastic mechanical properties(mechanical strength=191.5±14.98MPa),proper pore diameter(434.4±46.42?)in vitro and good biocompatibility in vivo.We further investigated the therapeutic effect of this novel scaffold on severe uterine injury(partial full-thickness defect of uterus)of 41 female SD rats.We randomly divided them into four groups: sham group,spontaneous regeneration group(Sr),simple scaffold group(Ss),3D printed scaffold group(Ps).Nighty days after implantation,rats which received 3D printed scaffolds showed evidently thicker endometrium regeneration(274.1±23.96?m,P<0.001)than endometrium in rats receiving simple scaffolds or in spontaneous regeneration group,it also obviously improved myometrium regeneration(smooth muscle content was 18.1%,P < 0.001),facilitated microvasculature regeneration(26±4.75,P=0.1306,difference was not statistically significant).Moreover,rats receiving 3D printed scaffolds restored the ability of endometrium for embryo implantation and development,and showed markedly higher pregnancy rates(12/16,P<0.001).These results indicated that this novel cell-laden bi-layer scaffold could be a promising strategy for endometrium regeneration in clinical.