Effect of four fluoroquinolones on the viability of bladder cancer cells in 2D and 3D cultures.

Introduction: The anticancer properties of fluoroquinolones and the high concentrations they achieve in urine may help in bladder cancer therapy. This study aimed to analyze the properties of 4 fluoroquinolones as potential candidates for supportive treatment of bladder cancer. Methods: Comparative analyses were performed on the cytotoxic effects of norfloxacin, enrofloxacin, moxifloxacin, and ofloxacin on normal and cancer urothelial cell lines. In 2D culture, the cytotoxic properties of fluoroquinolones were evaluated using MTT assay, real-time cell growth analysis, fluorescence and light microscopy, flow cytometry, and molecular analysis. In 3D culture, the properties of fluoroquinolones were tested using luminescence assays and confocal microscopy. Results and Discussion: All tested fluoroquinolones in 2D culture decreased the viability of both tested cell lines in a dose- and timedependent manner. Lower concentrations did not influence cell morphology and cytoskeletal organization. In higher concentrations, destruction of the actin cytoskeleton and shrinkage of the nucleus was visible. Flow cytometry analysis showed cell cycle inhibition of bladder cancer cell lines in the G2/M phase. This influence was minimal in the case of normal urothelium cells. In both tested cell lines, increases in the number of late apoptotic cells were observed. Molecular analysis showed variable expression of studied genes depending on the drug and concentration. In 3D culture, tested drugs were effective only in the highest tested concentrations which was accompanied by caspase 3/7 activation and cytoskeleton degradation. This effect was hardly visible in non-cancer cell lines. According to the data, norfloxacin and enrofloxacin had the most promising properties. These two fluoroquinolones exhibited the highest cytotoxic properties against both tested cell lines. In the case of norfloxacin, almost all calculated LC values for bladder cancer cell lines were achievable in the urine. Enrofloxacin and norfloxacin can be used to support chemotherapy in bladder cancer patients.

Quinolones as a Potential Drug in Genitourinary Cancer Treatment-A Literature Review.

Quinolones, broad-spectrum antibiotics, are frequently prescribed by urologists for many urological disorders. The mechanism of their bactericidal activity is based on the inhibition of topoisomerase II or IV complex with DNA, which consequently leads to cell death. It has been observed that these antibiotics also act against the analogous enzymes present in eukaryotic cells. Due to their higher accumulation in urine and prostate tissue than in serum, these drugs seem to be ideal candidates for application in genitourinary cancer treatment. In this study, an extensive literature review has been performed to collect information about concentrations achievable in urine and prostate tissue together with information about anticancer properties of 15 quinolones. Special attention was paid to the application of cytotoxic properties of quinolones for bladder and prostate cancer cell lines. Data available in the literature showed promising properties of quinolones, especially in the case of urinary bladder cancer treatment. In the case of prostate cancer, due to low concentrations of quinolones achievable in prostate tissue, combination therapy with other chemotherapeutics or another method of drug administration is necessary.

Urinary bladder augmentation with acellular biologic scaffold-A preclinical study in a large animal model.

Current strategies in urinary bladder augmentation include use of gastrointestinal segments, however, the technique is associated with inevitable complications. An acellular biologic scaffold seems to be a promising option for urinary bladder augmentation. The aim of this study was to evaluate the utility of bladder acellular matrix (BAM) for reconstruction of clinically significant large urinary bladder wall defects in a long-term porcine model. Urinary bladders were harvested from 10 pig donors. Biological scaffolds were prepared by chemically removing all cellular components from urinary bladder tissue. A total of 10 female pigs underwent hemicystectomy and subsequent bladder reconstruction with BAM. The follow-up study was 6 months. Reconstructed bladders were subjected to radiological, macroscopic, histological, immunohistochemical, and molecular evaluations. Six out of ten animals survived the 6-month follow-up period. Four pigs died during observation due to mechanical failure of the scaffold, anastomotic dehiscence between the scaffold and native bladder tissue, or occluded catheter. Tissue engineered bladder function was normal without any signs of postvoid residual urine in the bladder or upper urinary tracts. Macroscopically, graft shrinkage was observed. Urothelium completely covered the luminal surface of the graft. Smooth muscle regeneration was observed mainly in the peripheral graft region and gradually decreased toward the center of the graft. Expression of urothelial, smooth muscle, blood vessel, and nerve markers were lower in the reconstructed bladder wall compared to the native bladder. BAM seems to be a promising biomaterial for reconstruction of large urinary bladder wall defects. Further research on cell-seeded BAM to enhance urinary bladder regeneration is required.

Ciprofloxacin and Levofloxacin as Potential Drugs in Genitourinary Cancer Treatment-The Effect of Dose-Response on 2D and 3D Cell Cultures.

INTRODUCTION: Introducing new drugs for clinical application is a very difficult, long, drawn-out, and costly process, which is why drug repositioning is increasingly gaining in importance. The aim of this study was to analyze the cytotoxic properties of ciprofloxacin and levofloxacin on bladder and prostate cell lines in vitro. METHODS: Bladder and prostate cancer cell lines together with their non-malignant counterparts were used in this study. In order to evaluate the cytotoxic effect of both drugs on tested cell lines, MTT assay, real-time cell growth analysis, apoptosis detection, cell cycle changes, molecular analysis, and 3D cultures were examined. RESULTS: Both fluoroquinolones exhibited a toxic effect on all of the tested cell lines. In the case of non-malignant cell lines, the cytotoxic effect was weaker, which was especially pronounced in the bladder cell line. A comparison of both fluoroquinolones showed the advantage of ciprofloxacin (lower doses of drug caused a stronger cytotoxic effect). Both fluoroquinolones led to an increase in late apoptotic cells and an inhibition of cell cycle mainly in the S phase. Molecular analysis showed changes in BAX, BCL2, TP53, and CDKN1 expression in tested cell lines following incubation with ciprofloxacin and levofloxacin. The downregulation of topoisomerase II genes (TOP2A and TOP2B) was noticed. Three-dimensional (3D) cell culture analysis confirmed the higher cytotoxic effect of tested fluoroquinolone against cancer cell lines. CONCLUSIONS: Our results suggest that both ciprofloxacin and levofloxacin may have great potential, especially in the supportive therapy of bladder cancer treatment. Taking into account the low costs of such therapy, fluoroquinolones seem to be ideal candidates for repositioning into bladder cancer therapeutics.

Porphyrin Based 2D-MOF Structures as Dual-Kinetic Sorafenib Nanocarriers for Hepatoma Treatment.

The existing clinical protocols of hepatoma treatment require improvement of drug efficacy that can be achieved by harnessing nanomedicine. Porphyrin-based, paddle-wheel framework (PPF) structures were obtained and tested as dual-kinetic Sorafenib (SOR) nanocarriers against hepatoma. We experimentally proved that sloughing of PPF structures combined with gradual dissolving are effective mechanisms for releasing the drug from the nanocarrier. By controlling the PPF degradation and size of adsorbed SOR deposits, we were able to augment SOR anticancer effects, both in vitro and in vivo, due to the dual kinetic behavior of SOR@PPF. Obtained drug delivery systems with slow and fast release of SOR influenced effectively, although in a different way, the cancer cells proliferation (reflected with EC50 and ERK 1/2 phosphorylation level). The in vivo studies proved that fast-released SOR@PPF reduces the tumor size considerably, while the slow-released SOR@PPF much better prevents from lymph nodes involvement and distant metastases.

Increased Expression of p63 Protein and Sonic Hedgehog Signaling Molecule in Buccal Epithelial Holoclones.

Construction of many tissues and organs de novo requires the use of external epithelial cell sources. In the present study, we optimized the isolation, expansion, and characterization of porcine oral epithelial cells from buccal tissue (Buccal Epithelial Cells, BECs). Additionally, we tested whether key markers [cytokeratin 14 (ck14), p63 protein, and sonic hedgehog molecule (shh)] expression profiles are correlated with three buccal epithelial clone types. Two digestion methods of BECs isolation [Method 1, M1 (collagenase IV/dispase and accutase) and Method 2, M2 (collagenase IV/dispase and trypsin/EDTA)] were compared. Cells obtained by more effective method were further cultured to the third passage and analyzed. Holoclone-, meroclone-, and paraclone-like colonies were identified based on BEC morphology. Immunofluorescent staining was performed to compare selected markers for the indicated buccal clone types. Comparative analysis demonstrated the advantage of isolation using M1 over M2. Cells from the third passage exhibited average 92.73% ± 2.27% presence of ck14. Real-time polymerase chain reaction confirmed expression of tested genes [cytokeratin 8 (ck8), ck14, integrin ß1, and p63]. The highest level of ck14, shh and p63, was observed for holoclones. The comparable ck14 expression was observed in the mero- and paraclones. Meroclones expressed significantly lower levels of shh compared with paraclones. The weakest p63 expression was observed in the paraclone-like cells. It was demonstrated that holoclones are the richest in shh (+) and p63 (+) stem cells and these cells should appear to be a promising alternative for obtaining epithelial cells for tissue engineering purposes.

A tissue-engineered urinary conduit in a porcine urinary diversion model.

The use of an ileal segment is a standard method for urinary diversion after radical cystectomy. Unfortunately, utilization of this method can lead to numerous surgical and metabolic complications. This study aimed to assess the tissue-engineered artificial conduit for urinary diversion in a porcine model. Tissue-engineered tubular polypropylene mesh scaffolds were used for the right ureter incontinent urostomy model. Eighteen male pigs were divided into three equal groups: Group 1 (control ureterocutaneostomy), Group 2 (the right ureter-artificial conduit-skin anastomoses), and Group 3 (4 weeks before urostomy reconstruction, the artificial conduit was implanted between abdomen muscles). Follow-up was 6 months. Computed tomography, ultrasound examination, and pyelogram were used to confirm the patency of created diversions. Morphological and histological analyses were used to evaluate the tissue-engineered urinary diversion. All animals survived the experimental procedures and follow-up. The longest average patency was observed in the 3rd Group (15.8 weeks) compared to the 2nd Group (10 weeks) and the 1st Group (5.8 weeks). The implant's remnants created a retroperitoneal post-inflammation tunnel confirmed by computed tomography and histological evaluation, which constitutes urostomy. The simultaneous urinary diversion using a tissue-engineered scaffold connected directly with the skin is inappropriate for clinical application.

A new heterotropic vascularized model of total urinary bladder transplantation in a rat model.

This study developed a new procedure of urinary bladder transplantation on a rat model (n = 40). Heterotopic urinary bladder transplantation (n = 10) in the right groin vessels was performed. Direct urinary bladder examination, microangiography, histological analysis, and India ink injection were performed to evaluate the proposed method's functionality. Observation time was four weeks. One week after the procedure, the graft survival rate was 80%, two urinary bladders were lost due to anastomosis failure. The rest of the grafts survived two weeks without any complications. Lack of transitional epithelium or smooth muscle layer loss and lack of inflammatory process development were observed. This study was performed in order to obtain the necessary knowledge about urinary bladder transplantation. The proposed technique offers a new approach to the existing orthotropic models.

Biostimulative effect of laser on growth of mesenchymal stem/stromal cells in vitro.

INTRODUCTION: Human adipose tissue-derived mesenchymal stem/stromal cells (hAT-MSCs) are multipotent stromal cells with a high potential application in tissue engineering and regenerative medicine. Laser irradiation of the place where the cells were implanted can stimulate their proliferation, increase the secretion of growth factors and thus increase the therapeutic effect. AIM: To evaluate the influence of two lasers: Er:YAG and diode on the growth of hAT-MSCs in vitro. MATERIAL AND METHODS: hAT-MSCs were isolated from human subcutaneous adipose tissue. Immunophenotype of hAT-MSCs was confirmed by flow cytometry. Multipotency of hAT-MSCs was confirmed by differentiation into adipogenic, osteogenic and chondrogenic lineages. hAT-MSCs were irradiated with Er:YAG laser (wavelength 2940 nm, frequency 5, 10 Hz, doses: 0.1-1.2 J/cm2) for 2 s and 4 s and diode laser (wavelength 635 nm and doses: 1-8 J/cm2) for 5, 10, 20, 30 and 40 s. Cell viability was analysed 24 h after the exposure using MTT assay. RESULTS: Growth stimulation of hAT-MSCs after 5 Hz Er:YAG laser exposure, 0.1 J/cm2 dose for 4 s and 0.3 J/cm2 dose for 4 s was shown in comparison with the control group. Significant growth stimulation of hAT-MSCs after diode laser irradiation in doses of 1-4 J/cm2 was demonstrated compared to the control group. CONCLUSIONS: The presented results indicate that both lasers, Er:YAG and diode can be used to stimulate stem/stromal cell growth in vitro. The biostimulative effect of laser therapy on stromal cells may be used in the future in aesthetic dermatology in combined laser and cell therapy.

Molecular Aspects of Adipose-Derived Stromal Cell Senescence in a Long-Term Culture: A Potential Role of Inflammatory Pathways.

Long-term culture of mesenchymal stromal/stem cells in vitro leads to their senescence. It is very important to define the maximal passage to which the mesenchymal stromal/stem cells maintain their regenerative properties and can be used for cellular therapies and construction of neo-organs for clinical application. Adipose-derived stromal/stem cells were isolated from porcine adipose tissue. Immunophenotype, population doubling time, viability using bromodeoxyuridine assay, MTT assay, clonogencity, ß-galactosidase activity, specific senescence-associated gene expression, apoptosis, and cell cycle of adipose-derived mesenchymal stromal/stem cells (AD-MSCs) were analyzed. All analyses were performed through 12 passages (P). Decreasing viability and proliferative potential of AD-MSCs with subsequent passages together with prolonged population doubling time were observed. Expression of ß-galactosidase gradually increased after P6. Differentiation potential of AD-MSCs into adipogenic, chondrogenic, and osteogenic lineages decreased at the end of culture (P10). No changes in the cell cycle, the number of apoptotic cells and expression of specific AD-MSC markers during the long-term culture were revealed. Molecular analysis showed increased expression of genes involved in activation of inflammatory response. AD-MSCs can be cultured for in vivo applications without loss of their properties up to P6.

CD133 Antigen as a Potential Marker of Melanoma Stem Cells: In Vitro and In Vivo Studies.

Melanoma is the most dangerous type of skin cancer. Cancer stem cells (CSCs) are suspected to be responsible for the cancer recurrence and in the consequence for cancer therapy failure. CD133 is a potential marker for detection of melanoma CSCs. Experiments were performed on the B16-F10 mouse melanoma cell line. CD133+ cells were isolated using an immunomagnetic cell sorting technique. After isolation proliferative and clonogenic potential of CD133+, CD133- and CD133+/- were evaluated. The potential of CD133+ and CD133- cells for tumor induction was conducted on C57BL/6J mouse model. Three different cell quantities (100, 1000, 10000) were tested. Tumor morphology, number of mitoses, and tumor necrosis area were analyzed. Average 0.12% CD133+ cells were isolated. Compared to CD133- and unsorted CD133+/- cells, CD133+ cells were characterized by the higher proliferative and clonogenic potential. These properties were not confirmed in vivo, as both CD133+ and CD133- cells induced tumor growth in mouse model. No statistical differences in mitosis number and tumor necrosis area were observed. Simultaneous detection of CD133 antigen with other markers is necessary for accurate identification of these melanoma cancer stem cells.

The different expression of key markers on urothelial holoclonal, meroclonal, and paraclonal cells in in vitro culture.

Urothelial cell populations which differ in morphology and proliferation capacities can be isolated from the urinary bladder. The goal of this study was to analyze a clonal, proliferative, and self-renewing potential of porcine urothelial cells and to compare expression of selected adhesion and tight junction molecules, urothelial and stem cell markers for the urothelial clone types. Urothelial cells were isolated from 10 porcine urinary bladders. Three different clone types: holoclone-, meroclone-and paraclone-like colonies were identified based on their morphology. To characterize and compare the urothelial clones the immunofluorescent stains were performed. Expression of pancytokeratin (PanCK), Ki-67 and p63 was higher for holoclone- like cells compared to meroclone-and paraclone-like cells (P < 0.05). Meroclone-like cells expressed higher levels of p63 compared to paraclone- like cells (P < 0.05). The level of Ki-67 and PanCK for meroclone- and paraclone- like cells was comparable (P > 0.05). ß1 and ß4 integrins were not expressed. Expression of zonula occludens-1 (ZO-1) in cell-cell junctions for paraclone-, meroclone-and holoclone-like cells was 17.6 ± 0.6, 14.7 ± 0.5, and 16.1 ± 0.4, respectively. The results of actin filaments (F-actin) expression were 253,634 ± 6,920 for meroclone-like cells, 198,512 ± 7,977 for paraclone-like cells and 133,544 ± 3,169 for holoclone-like cells. Three urothelial cell types with differing features can be isolated from the bladder. Holoclone-like cells are the richest in stem cells and should be used in further studies for construction of neo-bladder or neo-conduit using tissue engineering methods.

Constructing artificial urinary conduits: current capabilities and future potential.

INTRODUCTION: Intestinal segments are currently used in reconstructive urology to create urinary diversion after cystectomy. Ileal conduit (IC) is the dominant type of urinary diversion. Nevertheless, IC is not an ideal solution as the procedure still requires entero-enterostomy to restore the bowel continuity. This step is a source of relevant complications that might prolong recovery time. Fabrication of artificial urinary conduit is a tempting idea to introduce an alternative form of urinary diversion which might improve cystectomy outcomes. AREAS COVERED: The aim of this review is to discuss available research data about artificial urinary conduit and identify major challenges for future studies. EXPERT OPINION: Fabrication of artificial urinary conduit is in range of current tissue engineering technology but there are still many challenges to overcome. There is an urgent need for studies to be conducted on large animal models with long follow up to expose the limitation of experimental strategies and to gather data for translational research.

Stem cells and differentiated cells differ in their sensitivity to urine in vitro.

Urinary tract regeneration using tissue engineering is one of the most challenging issues in the field of reconstructive urology. Cells seeded on scaffold are exposed to urine immediately after the implantation. The outcome of urinary bladder regeneration is depended on the ability of these cells to survive, proliferate, and regenerate. The aim of this study was to compare a sensitivity of three different cell lines to urine in vitro. Three different cell lines were isolated from porcine bladder (urothelial cells, UCs and smooth muscle cells, SMCs) and adipose tissue (adipose-derived stem cells, ADSCs). Cell viability (MTT assay), proliferation (real-time cell analysis using xCELLigence system) and apoptosis/necrosis (flow cytometry) were analyzed after exposition to urine. ADSCs were the most sensitive to urine compared to two other tested cell lines. Among the bladder cell lines the UCs were more resistant to urine than SMCs. Twenty four hour incubation of UCs, SMCs, and ADSCs with urine lead to ∼40%, ∼70%, and ∼90% reduction of their viability, respectively. The mechanism of urine mediated cytotoxicity differed depending on the tested cell type. Urothelial and SMCs seems to be more suitable for urinary bladder regeneration compared to mesenchymal stem cells, however, these cells have limited application especially in the case of urinary bladder cancer.

Nanoparticle as a novel tool in hyperthermic intraperitoneal and pressurized intraperitoneal aerosol chemotheprapy to treat patients with peritoneal carcinomatosis.

The treatment of peritoneal surface malignances has changed considerably over the last thirty years. Unfortunately, the palliative is the only current treatment for peritoneal carcinomatosis (PC). Two primary intraperitoneal chemotherapeutic methods are used. The first is combination of cytoreductive surgery (CRS) and Hyperthermic IntraPEritoneal Chemotherapy (HIPEC), which has become the gold standard for many cases of PC. The second is Pressurized IntraPeritoneal Aerosol Chemotheprapy (PIPAC), which is promising direction to minimally invasive as safedrug delivery. These methods were improved through multicenter studies and clinical trials that yield important insights and solutions. Major method development has been made through nanomedicine, specifically nanoparticles. Here, we are presenting the latest advances of nanoparticles and their application to precision diagnostics and improved treatment strategies for PC. These advances will likely develop both HIPEC and PIPAC methods that used for in vitro and in vivo studies. Several benefits of using nanoparticles will be discussed including: 1) Nanoparticles as drug delivery systems; 2) Nanoparticles and Near Infrred (NIR) Irradiation; 3) use of nanoparticles in perioperative diagnostic and individualized treatment planning; 4) use of nanoparticles as anticancer dressing's, hydrogels and as active beeds for optimal reccurence prevention; and 5) finally the curent in vitro and in vivo studies and clinical trials of nanoparticles. The current review highlighted use of nanoparticles as novel tools in improving drug delivery to be effective for treatment patients with peritoneal carcinomatosis.

Concise Review: Tissue Engineering of Urinary Bladder; We Still Have a Long Way to Go?

Regenerative medicine is a new branch of medicine based on tissue engineering technology. This rapidly developing field of science offers revolutionary treatment strategy aimed at urinary bladder regeneration. Despite many promising announcements of experimental urinary bladder reconstruction, there has been a lack in commercialization of therapies based on current investigations. This is due to numerous obstacles that are slowly being identified and precisely overcome. The goal of this review is to present the current status of research on urinary bladder regeneration and highlight further challenges that need to be gradually addressed. We put an emphasis on expectations of urologists that are awaiting tissue engineering based solutions in clinical practice. This review also presents a detailed characteristic of obstacles on the road to successful urinary bladder regeneration from urological clinician perspective. A defined interdisciplinary approach might help to accelerate planning transitional research tissue engineering focused on urinary tracts. Stem Cells Translational Medicine 2017;6:2033-2043.

Are agricultural and natural sources of bio-products important for modern regenerative medicine? A review.

[b] Abstract Introduction and objectives[/b]. As tissue engineering and regenerative medicine have continued to evolve within the field of biomedicine, the fundamental importance of bio-products has become increasingly apparent. This true not only in cases where they are derived directly from the natural environment, but also when animals and plants are specially bred and cultivated for their production. [b]Objective.[/b] The study aims to present and assess the global influence and importance of selected bio-products in current regenerative medicine via a broad review of the existing literature. In particular, attention is paid to the matrices, substances and grafts created from plants and animals which could potentially be used in experimental and clinical regeneration, or in reconstructive procedures. [b]Summary.[/b] Evolving trends in agriculture are likely to play a key role in the future development of a number of systemic and local medical procedures within tissue engineering and regenerative medicine. This is in addition to the use of bio-products derived from the natural environment which are found to deliver positive results in the treatment of prospective patients.

Vascularization Potential of Electrospun Poly(L-Lactide-co-Caprolactone) Scaffold: The Impact for Tissue Engineering.

BACKGROUND Electrospun nanofibers have widespread putative applications in the field of regenerative medicine and tissue engineering. When compared to naturally occurring collagen matrices, electrospun nanofiber scaffolds have two distinct advantages: they do not induce a foreign body reaction and they are not at risk for biological contamination. However, the exact substrate, structure, and production methods have yet to be defined. MATERIAL AND METHODS In the current study, tubular-shaped poly(L-lactide-co-caprolactone) (PLCL) constructs produced using electrospinning technology were evaluated for their potential application in the field of tissue regeneration in two separate anatomic locations: the skin and the abdomen. The constructs were designed to have an internal diameter of 3 mm and thickness of 200 µm. Using a rodent model, 20 PLCL tubular constructs were surgically implanted in the abdominal cavity and subcutaneously. The constructs were then evaluated histologically using electron microscopy at 6 weeks post-implantation. RESULTS Histological evaluation and analysis using scanning electron microscopy showed that pure scaffolds by themselves were able to induce angiogenesis after implantation in the rat model. Vascularization was observed in both tested groups; however, better results were obtained after intraperitoneal implantation. Formation of more and larger vessels that migrated inside the scaffold was observed after implantation into the peritoneum. In this group no evidence of inflammation and better integration of scaffold with host tissue were noticed. Subcutaneous implantation resulted in more fibrotic reaction, and differences in cell morphology were also observed between the two tested groups. CONCLUSIONS This study provides a standardized evaluation of a PLCL conduit structure in two different anatomic locations, demonstrating the excellent ability of the structure to achieve vascularization. Functional, histological, and mechanical data clearly indicate prospective clinical utilization of PLCL in critical size defect regeneration.

Does the Mesenchymal Stem Cell Source Influence Smooth Muscle Regeneration in Tissue-Engineered Urinary Bladders?

A variety of tissue engineering techniques utilizing different cells and biomaterials are currently being explored to construct urinary bladder walls de novo, but so far no approach is clearly superior. The aim of this study was to determine whether mesenchymal stem cells (MSCs) isolated from different sources, (bone marrow [BM-MSCs] and adipose tissue [ADSCs]), differ in their potential to regenerate smooth muscles in tissue-engineered urinary bladders and to determine an optimal number of MSCs for urinary bladder smooth muscle regeneration. Forty-eight rats underwent hemicystectomy and bladder augmentation with approximately 0.8 cm2 graft. In the first and second groups, urinary bladders were reconstructed with small intestinal submucosa (SIS) seeded with 10 × 106 or 4 × 106 ADSCs/cm2, respectively. In the third and fourth groups, urinary bladders were augmented with SIS seeded with 10 × 106 or 4 × 106 BM-MSCs/cm2, respectively. In the fifth group, urinary bladders were augmented with SIS without cells. The sixth group (control) was left intact. Smooth muscle regeneration was evaluated by real-time polymerase chain reaction (RT-PCR) and histological examinations. Histologically, there were no significant differences between urinary bladders augmented with ADSCs and BM-MSCs, but there was a marked increase in smooth muscle formation in bladders augmented with grafts seeded with MSCs in higher density (10 × 106/cm2) compared to lower density (4 × 106/cm2). Molecular analysis revealed that bladders reconstructed with ADSC-seeded grafts expressed higher levels of smooth muscle myosin heavy chain, caldesmon, and vinculin. Bladders augmented with unseeded SIS were fibrotic and devoid of smooth muscles. ADSCs and BM-MSCs have comparable smooth muscle regenerative potential, but the number of MSCs used for graft preparation significantly affects the smooth muscle content in tissue-engineered urinary bladders.