AbstractBackground:Traditional tissue engineering methods to fabricate urinary tract patch have some drawbacks such as compromised cell viability and uneven cell distribution within scaffold. In this study, we combined three-dimensional (3D) bioprinting and tissue engineering method to form a tissue-engineered urinary tract patch, which could be employed for the application on Beagles urinary tract defect mode to verify its effectiveness on urinary tract reconstruction.Methods:Human adipose-derived stem cells (hADSCs) were dropped into smooth muscle differentiation medium to generate induced microtissues (ID-MTs), flow cytometry was utilized to detect the positive percentage for CD44, CD105, CD45, and CD34 of hADSCs. Expression of vascular endothelial growth factor A (VEGFA) and tumor necrosis factor-stimulated gene-6 (TSG-6) in hADSCs and MTs were identified by Western blotting. Then the ID-MTs were employed for 3D bioprinting. The bioprinted structure was encapsulated by transplantation into the subcutaneous tissue of nude mice for 1 week. After retrieval of the encapsulated structure, hematoxylin and eosin and Masson’s trichrome staining were performed to demonstrate the morphology and reveal collagen and smooth muscle fibers, integral optical density (IOD) and area of interest were calculated for further semiquantitative analysis. Immunofluorescent double staining of CD31 and α-smooth muscle actin (α-SMA) were used to reveal vascularization of the encapsulated structure. Immunohistochemistry was performed to evaluate the expression of interleukin-2 (IL-2), α-SMA, and smoothelin of the MTs in the implanted structure. Afterward, the encapsulated structure was seeded with human urothelial cells. Immunofluorescent staining of cytokeratins AE1/AE3 was applied to inspect the morphology of seeded encapsulated structure.Results:The semi-quantitative assay showed that the relative protein expression of VEGFA was 0.355 ± 0.038 in the hADSCs vs. 0.649 ± 0.150 in the MTs (t = 3.291, P = 0.030), while TSG-6 expression was 0.492 ± 0.092 in the hADSCs vs. 1.256 ± 0.401 in the MTs (t = 3.216, P = 0.032). The semi-quantitative analysis showed that the mean IOD of IL-2 in the MT group was 7.67 ± 1.26, while 12.6 ± 4.79 in the hADSCs group, but semi-quantitative analysis showed that there was no statistical significance in the difference between the two groups (t = 1.724, P = 0.16). The semi-quantitative analysis showed that IOD was 71.7 ± 14.2 in non-induced MTs (NI-MTs) vs. 35.7 ± 11.4 in ID-MTs for collagen fibers (t = 3.428, P = 0.027) and 12.8 ± 1.9 in NI-MTs vs. 30.6 ± 8.9 in ID-MTs for smooth muscle fibers (t= 3.369, P = 0.028); furthermore, the mean IOD was 0.0613 ± 0.0172 in ID-MTs vs. 0.0017 ± 0.0009 in NI-MTs for α-SMA (t = 5.994, P = 0.027), while 0.0355 ± 0.0128 in ID-MTs vs. 0.0035 ± 0.0022 in NI-MTs for smoothelin (t= 4.268, P = 0.013), which indicate that 3D bioprinted structure containing ID-MTs could mimic the smooth muscle layer of native urinary tract. After encapsulation of the urinary tract patch for additional cell adhesion, urothelial cells were seeded onto the encapsulated structures, and a monolayer urothelial cell was observed.Conclusion:Through 3D bioprinting and tissue engineering methods, we provided a promising way to fabricate tissue-engineered urinary tract patch for further investigation.
