Stable laminar shear stress induces G1 cell cycle arrest and autophagy in urothelial carcinoma by a torque sensor-coupled cone-and-plate device.

The microenvironments of urinary systems play crucial roles in the development and metastasis of cancers due to their generation of complex temporal and spatial fluidic profiles. Because of their versatility in creating desired biomimetic flow, cone-and-plate bioreactors offer great potential for bladder cancer research. In this study, we construct a biocompatible cone-and-plate device coupled with a torque sensor, enabling the application and real-time monitoring of stable shear stress up to 50 dyne/cm². Under a stable shear stress stimulation at 12 dyne/cm2, bladder cancer cell BFTC-905 is arrested at the G1 phase with decreased cell proliferation after 24-hour treatment. This effect is associated with increased cyclin-dependent kinase inhibitors p21 and p27, inhibiting cyclin D1/CDK4 complex with dephosphorylation of serine 608 on the retinoblastoma protein. Consequently, an increase in cyclin D3 and decreases in cyclin A2 and cyclin E2 are observed. Moreover, we demonstrate that the shear stress stimulation upregulates the expression of autophagy-related proteins Beclin-1, LC3B-I and LC3B-II, while caspase cleavages are not activated under the same condition. The design of this system and its application shed new light on flow-induced phenomena in the study of urothelial carcinomas.

Simulation of Soluble and Bound VEGF-stimulated in vitro Capillary-like Network Formation on Deformed Substrate.

Capillary plexus cultivation is crucial in tissue engineering and regenerative medicine. Theoretical simulations have been conducted to supplement the expensive experimental works. However, the mechanisms connecting mechanical and chemical stimuli remained undefined, and the functions of the different VEGF forms in the culture environment were still unclear. In this paper, we developed a hybrid model for simulating short-term in vitro capillary incubations. We used the Cellular Potts model to predict individual cell migration, morphology change, and continuum mechanics to quantify biogel deformation and VEGF transport dynamics. By bridging the mechanical regulation and chemical stimulation in the model, the results showed good agreement between the predicted network topology and experiments, in which elongated cells connected, forming the network cords and round cells gathered, creating cobblestone-like aggregates. The results revealed that the capillary-like networks could develop in high integrity only when the mechanical and chemical couplings worked adequately, with the cell morphology and haptotaxis driven by the soluble and bound forms of VEGF, respectively, functioning simultaneously.

Hydrostatic pressure facilitates calcium deposition and osteogenic gene expression in the osteoblastic differentiation of placenta-derived multipotent cells.

OBJECTIVE: We tested the osteoblastic differentiation effects caused by physical stimulation such as hydrostatic pressure using placenta-derived multipotent cells. MATERIALS AND METHODS: The placenta-derived multipotent cells (PDMCs) were treated with osteogenic medium to induce PDMCs differentiation into osteoblast-like cells. The induced PDMCs were stimulated using hydrostatic pressure at a magnitude of 30 kPa for 1 h/day for up to 12 days. The calcium deposition monitored by Alizarin Red staining and the calcium content of each experimental group were quantified. RESULTS: The results demonstrated both the calcium deposition and concentration were elevated through hydrostatic pressure stimulation. Moreover, in order to indicate of PDMC osteodifferentiation, RT-qPCR analysis were performed and mRNA expression of osteoblast differentiation markers (type I collagen, alkaline phosphatase, RUNX2, and BGLAP), the bone morphogenetic protein family (BMP1-7) and BMP receptors (BMPR1A, BMPR1B, and BMPR2) were examined. Among them, the mRNA levels of RUNX2, COL1A1, BMP1, BMP3, and BMPR1A increased significantly in the hydrostatic-pressure-stimulated groups, whereas BGLAP, ALP, BMP2, BMP6, BMPR1B, and BMPR2 exhibited a slight upregulation between the control and experimental groups, indicating the specific signal route induced by hydrostatic pressure on PDMCs. CONCLUSION: Our results revealed the beneficial effects of stem cells stimulated using hydrostatic pressure, which could enhance calcium deposition considerably and facilitate osteodifferentiation, and the results may be applied to tissue regeneration in the near future.

Involvement of L-type Ca²âº channel and toll-like receptor-4 in nickel-induced interleukin-8 gene expression.

The metal nickel (Ni(2+)) is found everywhere in our daily lives, including coins, costume jewelry, and even nuts and chocolates. Nickel poisoning can cause inflammatory reactions, respiratory diseases, and allergic contact dermatitis. To clarify the mechanism by which nickel induces mediators of inflammation, we used the human acute monocytic leukemia THP-1 cell line as a model. Interleukin (IL)-8 promoter activity as well as gene expression were tested by luciferase assay and real-time polymerase chain reaction. The underlying mechanisms of nickel-induced IL-8 were investigated. We found that nickel induced IL-8 gene expression via the L-type Ca(2+) channel, Toll-like receptor-4 (TRL-4) and nuclear factor NF-κB signal transduction pathways. Nickel activated NF-κB expression through extracellular signal-regulated kinase 1/2 phosphorylation and then increased IL-8 expression. Thus, the L-type Ca(2+) channel and TRL-4 play important roles in nickel-induced inflammatory gene expressions.

Osteogenic differentiation of placenta-derived multipotent cells in vitro.

OBJECTIVE: Stem cells offer great potential for clinical therapeutic use because of their ability to rejuvenate and to differentiate into numerous types of cells. We isolated multipotent cells from the human term placenta that were capable of differentiation into cells of all three germ layers. MATERIALS AND METHODS: We examined the ability of these placenta-derived multipotent cells (PDMCs) to differentiate into osteoblasts (OBs) or OB-like cells. The PDMCs were treated with osteogenic medium (OM) consisting of dexamethasone, ß-glycerol phosphate, and ascorbic acid. At sequential time intervals (0 day, 3 days, 6 days, 9 days, and 12 days) we measured several parameters. These included alkaline phosphatase (ALP) activity, alizarin red staining (ARS) to measure calcium deposition, messenger RNA (mRNA) expressions of osteogenesis-related transcription factor (Cbfa1), and calcium coordination protein (osteocalcin). These variables were used as indicators of PDMC osteodifferentiation. RESULTS: We showed that ALP activity in the early stage of differentiation and calcium deposition were both significantly increased in PDMCs after OM induction. Moreover, the Cbfa1 and osteocalcin gene expressions were upregulated. The results suggested that OM induced an osteodifferentiation potential in PDMCs. CONCLUSION: PDMC-derived osteocytes provide a useful model to evaluate the mechanisms of key biomolecules and bioengineering processes.

Toll-like receptor 6 and connective tissue growth factor are significantly upregulated in mitomycin-C-treated urothelial carcinoma cells under hydrostatic pressure stimulation.

BACKGROUND: Urothelial carcinoma (UC) is the most common histologic subtype of bladder cancer. The administration of mitomycin C (MMC) into the bladder after transurethral resection of the bladder tumor (TURBT) is a common treatment strategy for preventing recurrence after surgery. We previously applied hydrostatic pressure combined with MMC in UC cells and found that hydrostatic pressure synergistically enhanced MMC-induced UC cell apoptosis through the Fas/FasL pathways. To understand the alteration of gene expressions in UC cells caused by hydrostatic pressure and MMC, oligonucleotide microarray was used to explore all the differentially expressed genes. RESULTS: After bioinformatics analysis and gene annotation, Toll-like receptor 6 (TLR6) and connective tissue growth factor (CTGF) showed significant upregulation among altered genes, and their gene and protein expressions with each treatment of UC cells were validated by quantitative real-time PCR and immunoblotting. CONCLUSION: Under treatment with MMC and hydrostatic pressure, UC cells showed increasing apoptosis using extrinsic pathways through upregulation of TLR6 and CTGF.

Electrical stimulation to promote osteogenesis using conductive polypyrrole films.

In this study, we developed an electrical cell culture and monitoring device. Polypyrrole (PPy) films with different resistances were fabricated as conductive surfaces to investigate the effect of substrate-mediated electrical stimulation. The physical and chemical properties of the devices, as well as their biocompatibilities, were thoroughly evaluated. These PPy films had a dark but transparent appearance, on which the surface cells could be easily observed. After treating with the osteogenic medium, rat bone marrow stromal cells cultured on the PPy films differentiated into osteoblasts. The cells grown on the PPy films had up-regulated osteogenic markers, and an alkaline phosphatase activity assay showed that the PPy films accelerated cell differentiation. Alizarin red staining and calcium analysis suggested that the PPy films promoted osteogenesis. Finally, PPy films were subjected to a constant electric field to elucidate the effect of electrical stimulation on osteogenesis. Compared with the untreated group, electrical stimulation improved calcium deposition in the extracellular matrix. Furthermore, PPy films with lower resistances allowed larger currents to stimulate the surface cells, which resulted in higher levels of mineralization. Overall, these results indicated that this system exhibited superior electroactivity with controllable electrical resistance and that it can be coated directly to produce medical devices with a transparent appearance, which should be beneficial for research on electrical stimulation for tissue regeneration.

Hydrostatic pressure enhances mitomycin C induced apoptosis in urothelial carcinoma cells.

OBJECTIVES: Urothelial carcinoma (UC) of the bladder is the second most common cancer of the genitourinary system. Clinical UC treatment usually involves transurethral resection of the bladder tumor followed by adjuvant intravesical immunotherapy or chemotherapy to prevent recurrence. Intravesical chemotherapy induces fewer side effects than immunotherapy but is less effective at preventing tumor recurrence. Improvement to intravesical chemotherapy is, therefore, needed. METHODS AND MATERIALS: Cellular effects of mitomycin C (MMC) and hydrostatic pressure on UC BFTC905 cells were assessed. The viability of the UC cells was determined using cellular proliferation assay. Changes in apoptotic function were evaluated by caspase 3/7 activities, expression of FasL, and loss of mitochondrial membrane potential. RESULTS: Reduced cell viability was associated with increasing hydrostatic pressure. Caspase 3/7 activities were increased following treatment of the UC cells with MMC or hydrostatic pressure. In combination with 10 kPa hydrostatic pressure, MMC treatment induced increasing FasL expression. The mitochondria of UC cells displayed increasingly impaired membrane potentials following a combined treatment with 10 µg/ml MMC and 10 kPa hydrostatic pressure. CONCLUSIONS: Both MMC and hydrostatic pressure can induce apoptosis in UC cells through an extrinsic pathway. Hydrostatic pressure specifically increases MMC-induced apoptosis and might minimize the side effects of the chemotherapy by reducing the concentration of the chemical agent. This study provides a new and alternative approach for treatment of patients with UC following transurethral resection of the bladder tumor.

Use of biotinylated chitosan for substrate-mediated gene delivery.

To improve transfection efficiency of nonviral vectors, biotinylated chitosan was applied to complex with DNA in different N/P ratios. The morphologies and the sizes of formed nanoparticles were suitable for cell uptake. The biotinylation decreased the surface charges of nanoparticles and hence reduced the cytotoxicity. The loading capacities of chitosan were slightly decreased with the increase of biotinylation, but most of the DNA molecules were still complexed. Using different avidin-coated surfaces, the interaction between biotinylated nanoparticles to the substrate may be manipulated. The in vitro transfection results demonstrated that biotinylated nanoparticles may be bound to avidin coated surfaces, and the transfection efficiencies were thus increased. Through regulating the N/P ratio, biotinylation levels, and surface avidin, the gene delivery can be optimized. Compared to the nonmodified chitosan, biotinylated nanoparticles on biomaterial surfaces can increase their chances to contact adhered cells. This spatially controlled gene delivery improved the gene transfer efficiency of nonviral vectors and could be broadly applied to different biomaterial scaffolds for tissue engineering applications.