Encapsulated Three-Dimensional Bioprinted Structure Seeded with Urothelial Cells: A New Construction Technique for Tissue-Engineered Urinary Tract Patch
The reconstruction of the urinary tract remains a significant challenge in urology, particularly due to the lack of suitable biomaterials for replacing damaged or defective tissues. Traditional methods of urinary tract reconstruction often rely on autografts, such as buccal mucosa or prepuce, which, while effective, come with limitations including donor site morbidity and limited availability. This has driven researchers to explore tissue engineering as a viable alternative. However, conventional tissue engineering approaches face challenges such as compromised cell viability, uneven cell distribution, and prolonged preparation times. In this study, we present a novel technique that combines three-dimensional (3D) bioprinting and tissue engineering to fabricate a urinary tract patch, addressing these limitations and offering a promising solution for urinary tract reconstruction.
Background and Motivation
Urinary tract strictures, often caused by catheterization, trauma, or infection, frequently require surgical intervention. The lack of autologous materials has led to the exploration of natural and synthetic biomaterials for urinary tract reconstruction. Natural biomaterials, such as small intestinal submucosa and bladder acellular matrix (BAM), offer biocompatibility but face issues like disease transmission and heterogeneity. Synthetic biomaterials, such as polyglycolic acid and poly lactic-co-glycolic acid (PLGA), provide structural support but lack molecular signals for cell attachment, leading to compromised cell activity and proliferation. Both types of biomaterials struggle to achieve spatial cell distribution, which is crucial for tissue function.
Traditional tissue engineering methods involve seeding cells onto porous biomaterials and waiting for cell infiltration, a process that can take weeks. This prolonged preparation time makes it difficult to produce “off the shelf” tissue-engineered patches for clinical use. 3D bioprinting, an emerging technology derived from additive manufacturing, offers a solution by enabling the construction of scaffolds containing cells in a single step. This method allows for precise control over cell distribution and scaffold shape, overcoming the limitations of traditional approaches.
Methods
Cell Culture and Characterization
Human adipose-derived stem cells (hADSCs) were cultured and characterized using flow cytometry to confirm their mesenchymal stem cell phenotype. The cells were positive for CD44 and CD105, markers of mesenchymal stem cells, and negative for CD45 and CD34, markers of hematopoietic cells. The hADSCs were then induced to differentiate into smooth muscle-like cells using a smooth muscle differentiation medium containing transforming growth factor-beta1 (TGF-β1). The resulting microtissues (MTs) were compared to non-induced microtissues (NI-MTs) in terms of protein expression and differentiation potential.
3D Bioprinting
The induced microtissues (ID-MTs) were embedded in a hydrogel composed of gelatin and sodium alginate and bioprinted using a micro-extrusion 3D bioprinter. The bioprinted structures were cross-linked with calcium chloride and cultured in differentiation medium. The bioprinted structures were then implanted subcutaneously into nude mice for one week to allow for encapsulation and maturation.
Histological and Immunofluorescent Analysis
After retrieval, the encapsulated structures were analyzed using hematoxylin and eosin (H&E) staining, Masson’s trichrome staining, and immunofluorescent staining. H&E staining revealed the morphology of the MTs and hADSCs within the bioprinted structures. Masson’s trichrome staining was used to identify collagen and smooth muscle fibers. Immunofluorescent staining of CD31 and alpha-smooth muscle actin (α-SMA) was performed to assess vascularization, while immunohistochemistry (IHC) was used to evaluate the expression of smooth muscle markers (α-SMA and smoothelin) and interleukin-2 (IL-2).
Urothelial Cell Seeding
Human urothelial cells (UCs) were isolated from ureteral tissues and cultured. The UCs were then seeded onto the encapsulated bioprinted structures and cultured for one week. The morphology and distribution of the UCs on the structures were assessed using immunofluorescent staining of cytokeratins AE1/AE3.
Results
Protein Expression in hADSCs and MTs
Western blot analysis revealed that the expression of vascular endothelial growth factor A (VEGFA) and tumor necrosis factor-stimulated gene-6 (TSG-6) was significantly higher in MTs compared to monolayer-cultured hADSCs. The relative protein expression of VEGFA was 0.355 ± 0.038 in hADSCs vs. 0.649 ± 0.150 in MTs (t = 3.291, P = 0.030), while TSG-6 expression was 0.492 ± 0.092 in hADSCs vs. 1.256 ± 0.401 in MTs (t = 3.216, P = 0.032). These results suggest that MTs have enhanced potential for vascularization and anti-inflammatory effects compared to single hADSCs.
Histological and Immunofluorescent Findings
H&E staining showed that the MTs maintained their morphology after bioprinting and subcutaneous implantation, while hADSCs adopted a round shape. Masson’s trichrome staining revealed that ID-MTs produced both collagen and smooth muscle fibers, while NI-MTs primarily produced collagen fibers. The mean integral optical density (IOD) for collagen fibers was 71.7 ± 14.2 in NI-MTs vs. 35.7 ± 11.4 in ID-MTs (t = 3.428, P = 0.027), while the IOD for smooth muscle fibers was 12.8 ± 1.9 in NI-MTs vs. 30.6 ± 8.9 in ID-MTs (t = 3.369, P = 0.028). These results indicate that ID-MTs can mimic the smooth muscle layer of the native urinary tract.
Immunofluorescent staining of CD31 and α-SMA demonstrated neo-vascularization in the MT group, with CD31+ cells localized around α-SMA-positive vascular-like structures. In contrast, the hADSC group showed minimal vascularization. IHC staining revealed that the expression of α-SMA and smoothelin was significantly higher in ID-MTs compared to NI-MTs, confirming the maintenance of smooth muscle differentiation in ID-MTs after implantation.
Urothelial Cell Seeding and Morphology
UCs seeded onto the encapsulated bioprinted structures formed a monolayer on the surface, as confirmed by immunofluorescent staining of cytokeratins AE1/AE3. This suggests that the bioprinted structures can support the adhesion and growth of urothelial cells, which are essential for creating a functional barrier in the urinary tract.
Discussion
The results of this study demonstrate the potential of combining 3D bioprinting and tissue engineering to create a urinary tract patch. The use of ID-MTs composed of hADSCs offers several advantages over traditional methods, including enhanced cell viability, improved vascularization, and the ability to mimic the smooth muscle layer of the native urinary tract. The higher expression of VEGFA and TSG-6 in MTs compared to monolayer-cultured hADSCs suggests that MTs can promote vascularization and reduce inflammation, which are critical for the success of tissue-engineered constructs.
The ability of ID-MTs to maintain their smooth muscle phenotype after implantation is particularly noteworthy, as it addresses a common challenge in tissue engineering: the loss of cell differentiation during scaffold integration. The successful seeding of urothelial cells onto the encapsulated bioprinted structures further supports the feasibility of this approach for urinary tract reconstruction.
Conclusion
This study presents a novel technique for fabricating a tissue-engineered urinary tract patch using 3D bioprinting and encapsulated MTs. The results demonstrate that ID-MTs composed of hADSCs can mimic the smooth muscle layer of the native urinary tract, promote vascularization, and support the adhesion of urothelial cells. This approach offers a promising solution for urinary tract reconstruction and warrants further investigation in animal models to assess its effectiveness in vivo.
doi.org/10.1097/CM9.0000000000000654
Was this helpful?
0 / 0