Kaile Zhang

3D bioprinting of urethra with PCL/PLCL blend and dual autologous cells in fibrin hydrogel: An in vitro evaluation of biomimetic mechanical property and cell growth environment

OBJECTIVE Urethral stricture is a common condition seen after urethral injury. The currently available treatments are inadequate and there is a scarcity of substitute materials used for treatment of urethral stricture. The traditional tissue engineering of urethra involves scaffold design, fabrication and processing of multiple cell types. METHODS In this study, we have used 3D bioprinting technology to fabricate cell-laden urethra in vitro with different polymer types and structural characteristics. We hypothesized that use of PCL and PLCL polymers with a spiral scaffold design could mimic the structure and mechanical properties of natural urethra of rabbits, and cell-laden fibrin hydrogel could give a better microenvironment for cell growth. With using an integrated bioprinting system, tubular scaffold was formed with the biomaterials; meanwhile, urothelial cells (UCs) and smooth muscle cells (SMCs) were delivered evenly into inner and outer layers of the scaffold separately within the cell-laden hydrogel. RESULTS The PCL/PLCL (50:50) spiral scaffold demonstrated mechanical properties equivalent to the native urethra in rabbit. Evaluation of the cell bioactivity in the bioprinted urethra revealed that UCs and SMCs maintained more than 80% viability even at 7days after printing. Both cell types also showed active proliferation and maintained the specific biomarkers in the cell-laden hydrogel. CONCLUSION These results provided a foundation for further studies in 3D bioprinting of urethral constructs that mimic the natural urethral tissue in mechanical properties and cell bioactivity, as well a possibility of using the bioprinted construct for in vivo study of urethral implantation in animal model. SIGNIFICANCE OF STATEMENTS The 3D bioprinting is a new technique to replace traditional tissue engineering. The present study is the first demonstration that it is feasible to create a urethral construct. Two kinds of biomaterials were used and achieved mechanical properties equivalent to that of native rabbit urethra. Bladder epithelial cells and smooth muscle cells were loaded in hydrogel and maintained sufficient viability and proliferation in the hydrogel. The highly porous scaffold could mimic a natural urethral base-membrane, and facilitate contacts between the printed epithelial cells and smooth muscle cells on both sides of the scaffold. These results provided a strong foundation for future studies on 3D bioprinted urethra.

Application of Wnt Pathway Inhibitor Delivering Scaffold for Inhibiting Fibrosis in Urethra Strictures: In Vitro and in Vivo Study

Objective: To evaluate the mechanical property and biocompatibility of the Wnt pathway inhibitor (ICG-001) delivering collagen/poly(l-lactide-co-caprolactone) (P(LLA-CL)) scaffold for urethroplasty, and also the feasibility of inhibiting the extracellular matrix (ECM) expression in vitro and in vivo. Methods: ICG-001 (1 mg (2 mM)) was loaded into a (P(LLA-CL)) scaffold with the co-axial electrospinning technique. The characteristics of the mechanical property and drug release fashion of scaffolds were tested with a mechanical testing machine (Instron) and high-performance liquid chromatography (HPLC). Rabbit bladder epithelial cells and the dermal fibroblasts were isolated by enzymatic digestion method. (3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide (MTT) assay) and scanning electron microscopy (SEM) were used to evaluate the viability and proliferation of the cells on the scaffolds. Fibrolasts treated with TGF-β1 and ICG-001 released medium from scaffolds were used to evaluate the anti-fibrosis effect through immunofluorescence, real time PCR and western blot. Urethrography and histology were used to evaluate the efficacy of urethral implantation. Results: The scaffold delivering ICG-001 was fabricated, the fiber diameter and mechanical strength of scaffolds with inhibitor were comparable with the non-drug scaffold. The SEM and MTT assay showed no toxic effect of ICG-001 to the proliferation of epithelial cells on the collagen/P(LLA-CL) scaffold with ICG-001. After treatment with culture medium released from the drug-delivering scaffold, the expression of Collagen type 1, 3 and fibronectin of fibroblasts could be inhibited significantly at the mRNA and protein levels. In the results of urethrography, urethral strictures and fistulas were found in the rabbits treated with non-ICG-001 delivering scaffolds, but all the rabbits treated with ICG-001-delivering scaffolds showed wide caliber in urethras. Histology results showed less collagen but more smooth muscle and thicker epithelium in urethras repaired with ICG-001 delivering scaffolds. Conclusion: After loading with the Wnt signal pathway inhibitor ICG-001, the Collagen/P(LLA-CL) scaffold could facilitate a decrease in the ECM deposition of fibroblasts. The ICG-001 delivering Collagen/P(LLA-CL) nanofibrous scaffold seeded with epithelial cells has the potential to be a promising substitute material for urethroplasty. Longer follow-up study in larger animals is needed in the future.

Current Stem Cell Biomarkers and Their Functional Mechanisms in Prostate Cancer

Currently there is little effective treatment available for castration resistant prostate cancer, which is responsible for the majority of prostate cancer related deaths. Emerging evidence suggested that cancer stem cells might play an important role in resistance to traditional cancer therapies, and the studies of cancer stem cells (including specific isolation and targeting on those cells) might benefit the discovery of novel treatment of prostate cancer, especially castration resistant disease. In this review, we summarized major biomarkers for prostate cancer stem cells, as well as their functional mechanisms and potential application in clinical diagnosis and treatment of patients.

Stem Cell Therapy for Treatment of Stress Urinary Incontinence: The Current Status and Challenges

Stress urinary incontinence (SUI) is a common urinary system disease that mostly affects women. Current treatments still do not solve the critical problem of urethral sphincter dysfunction. In recent years, there have been major developments in techniques to obtain, culture, and characterize autologous stem cells as well as many studies describing their applications for the treatment of SUI. In this paper, we review recent publications and clinical trials investigating the applications of several stem cell types as potential treatments for SUI and the underlying challenges of such therapy.

Electrospun nanoyarn seeded with myoblasts induced from placental stem cells for the application of stress urinary incontinence sling: An in vitro study.

OBJECTIVE To fabricate a novel electrospun nanoyarn using dynamic liquid electrospinning technique. The nanoyarn will be seeded with myoblasts differentiated from placental stem cells (PSCs) to evaluate the feasiblity of the cell-scaffold construct as a tissue engineering sling to treat stress urinary incontinence. MATERIAL AND METHODS PSCs were induced to myoblasts with 5-azacytidine and horse serum. Myoblasts differentiation was confirmed by immunofluorescence and western blot. Western blot was also used to assess the change of extracellular matrix (ECM) expression. A dynamic liquid electrospinning system was used to fabricate a novel nanoyarn scaffold for myoblast seeding. Cell morphology and proliferation on nanoyarn and nanofiber scaffold were compared with scanning electron microscopy (SEM) and MTS assay respectively. Filament actin development was tested with Rhodamine-labeled phalloidin stainning; cell infiltration into scaffolds was observed with H&E stainning. ECM expression was evaluated by a collagen assay, immunofluorescence imaging and real-time PCR. RESULTS Myoblasts showed increased expression of α-SMA, desmin, and collagen type 1, 3 when compared to PSCs. The nanoyarn possessed higher porosity, larger pore size, and aligned fibers/yarns as compared to nanofiber scaffold. Cell proliferation was significantly improved on nanoyarn scaffold. Cells could infiltrate deeply in the nanoyarn scaffold after 7 days in culture, however, they could only proliferate on the surface of the nanofiber scaffold. The myoblast-nanoyarn constructs seemed to be more like a muscle tissue. The myoblasts spreading on the nanoyarn scaffold were visible with aligned actin filaments in the horizontal view, whereas myoblasts spreading on the nanofiber scaffold were visible with unaligned actin filaments. Nanoyarn myoblasts exhibited higher production and density of type 1, 3 collagen and elastin. CONCLUSIONS PSCs could be induced into myoblast and expressed elevated myogenic markers and ECM. PSCs are potential cell source for a tissue engineered sling. The novel electrospun nanoyarn scaffold showed potential for use as a sling for treatment of stress urinary incontinence. In vitro studies demonstrated that the nanoyarn scaffold could improve cell proliferation, muscular tissue development, and ECM expression compared to random nanofiber scaffolds. The combination of myoblasts and nanoyarn scaffold could be a promising tissue engineered sling for future in vivo studies.

Let-7i-5p regulation of cell morphology and migration through distinct signaling pathways in normal and pathogenic fibroblasts from urethra

Pelvic fracture urethral distraction defects (PFUDD) is a common disease that could severely affect patients’ life quality, yet little is known about the molecular mechanism associated with pathogenic fibrosis in PFUDD. In this study, we found that let-7i-5p could regulate different cellular events in normal and pathogenic fibroblasts through three distinct signaling pathways. Interestingly, those regulations are compromised during the translation from mRNA to protein, and partially based on pathogenic status of the fibroblasts. By analyzing the molecular mechanism associated with its function, we conclude that let-7i-5p plays an essential role in regulating cell shape and tissue elasticity, cell migration, cell morphology and cytoskeleton, and could serve as a potential target for clinical treatment of urethral stricture patients.

The fabrication of 3D surface scaffold of collagen/poly (L-lactide-co-caprolactone) with dynamic liquid system and its application in urinary incontinence treatment as a tissue engineered sub-urethral sling: In vitro and in vivo study

To fabricate a novel nanoyarn biomaterial via a dynamic liquid electrospinning system, and to simultaneously evaluate whether nanoyarn is capable of being applied as a urinary sling for future clinical transfer.

microRNA expression profiles of scar and normal tissue from patients with posterior urethral stricture caused by pelvic fracture urethral distraction defects

Pelvic fracture urethral distraction defect (PFUDD) seriously affects the quality of life of patients. At present, there are few effective drug treatments available for PFUDD-induced urethral stricture, which is associated with fibrosis and scar formation in urethra lumen. Emerging evidence suggests that microRNAs (miRNAs/miRs) may be involved in the regulation of fibrosis, and analysis of miRNA expression profiles in urethral scar and normal urethra tissues may therefore benefit the discovery of novel treatments for urethral stricture with micro invasive procedures. In the present study, miRNA sequencing and quantitative polymerase chain reaction (qPCR) validation using paired scar and normal tissues from patients with PFUDD, and functional analysis of the miRNAs involved in the fibrosis associated signaling pathway was performed. A total of 94 differentially expressed miRNAs were identified in the scar tissue of patients with PFUDD. Among them, 26 miRNAs had significantly altered expression in the scar tissue compared with the normal tissue from the same patient. qPCR validation confirmed that miR-129-5p was overexpressed in scar tissue. The TGF-β pathway-associated functions of a total of 5 miRNAs (hsa-miR-129-5p, hsa-miR-135a-5p, hsa-miR-363-3p, hsa-miR-6720-3p and hsa-miR-9-5p) were further analyzed, as well as their key molecular targets and functional mechanisms in signaling regulation. To conclude the miRNA sequencing indicated a significantly altered expression of hsa-miR-129-5p, hsa-miR-135a-5p, hsa-miR-363-3p, hsa-miR-6720-3p and hsa-miR-9-5p in patients with PFUDD. These miRNAs and their potential target genes were associated with fibrosis in several diseases, and the data from the present study may help explore potential miRNA targets for future precision treatments for urethral stricture.