Tissue Engineered Urethra Based On Three Dimensional Bioprinting

Background and objective:Urethral stricture caused by catheterization,pelvic trauma,or infection often requires surgical intervention such as urethral reconstruction to restore the continuity of urethra.The lack of available autologous materials such as buccal mucosa and prepuce drives researchers to seek substitute biomaterials through tissue engineering.Three-dimensional（3D）bioprinting holds great promise in tissue engineering due to its effectiveness in construction of a structure containing spatially distributed stem cells.But 3D bioprinting also retains some drawbacks such as compromised cell viability and proliferation during bioprinting process,which could handicap cell function within the bioprinted structure.Smooth muscle differentiated human adipose derived stem cells（hADSCs）provide a crucial stem cell source for urethra tissue engineering,but the induction of hADSCs for smooth muscle differentiation still has several issues to overcome,including a relatively long induction time,which limits access to abundant stem cells within a short period of time for further application.The objective of our study was to improve the efficiency of hADSC smooth muscle differentiation and cell viability of a 3D bioprinted structure.Next,we employed the induced hADSCs and 3D bioprinting to fabricate tissue engineered urethra for further investigation.Material and methods:Hanging-drop method was utilized to generate hADSC microtissues（MTs）in a smooth muscle inductive medium containing human transforming growth factor β1,then immunofluoresent staining and western blotting were employed to detect the expression of smooth muscle markers（α-SMA and Smoothelin）.Then the induced MTs and hADSCs were bioprinted onto a 3D structure,followed by Live/dead and cell count kit 8 assays.Then the bioprinted 3D structure was encapsulated by subcutaneous implantation into nude mice for 1 week,and seeded with human urothelial cells（hUCs）which were isolated from human ureter tissues after retrieval to build an artificial urethra.HE and massons1 trichrome staining were conducted to manifest the morphology of the artificial urethra,immunofluorescent staining was used for detection of neo-vascularization and urothelial marker,immunohistochemistry was performed for phenotype examination of incorporated MTs.Results:After 3 days of smooth muscle induction,the expression of a-smooth muscle actin and smoothelin was higher in microtissues than in their counterpart monolayer cultured hADSCs,as confirmed by immunofluorescence and western blotting analysis.The semi-quantitative assay showed that the expression of a-smooth muscle actin（a-SMA）was 0.218±0.077 in MTs and 0.082±0.007 in Controls,P<0.001;smoothelin expression was 0.319±0.02 in MTs and 0.178±0₀6 in Controls,P<0.001.Cell viability and cell proliferation in the 3D structure printed with microtissues were higher at all time points compared to the conventional single-cell bioprinting strategy.Live/dead and cell count kit 8 assays showed that cell viability and cell proliferation in the 3D structure printed with microtissues were higher at all time points compared to the conventional single-cell bioprinting strategy（mean cell viability was 88.16%±3.98%vs.61,76%±15%for microtissues and single-cells,respectively,p=0.0396）.As for in vivo experiment,HE staining showed that after 1 week of implantation,the microtissues largely preserved their morphology,while hADSCs were turned into round shape,and Masson’s trichrome staining revealed that induced MTs were smooth muscle fiber-rich and collagen fiber-depleted.Immunohistochemistry demonstrated the expression of a-SMA and smoothelin in induced MTs and indicated the stability of their phenotypes.Moreover,immunofluorescence confirmed the neo-vascularization in the MTs group while the monolayer hADSC group only had little neo-vascularization,and monolayer hUCs were observed on the encapsulated structure by detection of urothelial marker.Conclusion:We combined hanging-drop method and 3D bioprinting to fabricate a 3D structure,and seeded hUCs with the encapsulated 3D strucuture harvested from nude mice subcutaneous implantation to build an artificial urethra,and analysed cell viability and phenotype within the artificial urethra,thus providing a novel way for urethra reconstruction in animal model of urethra defect.