Browsing by Subject "MC3T3-E1"

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    Rac1 and Cdc42 GTPases regulate shear stress-driven β-catenin signaling in osteoblasts
    (Elsevier, 2013) Wan, Qiaoqiao; Cho, Eunhye; Yokota, Hiroki; Na, Sungsoo; Anatomy, Cell Biology and Physiology, School of Medicine
    Beta-catenin-dependent TCF/LEF (T-cell factor/lymphocyte enhancing factor) is known to be mechanosensitive and an important regulator for promoting bone formation. However, the functional connection between TCF/LEF activity and Rho family GTPases is not well understood in osteoblasts. Herein we investigated the molecular mechanisms underlying oscillatory shear stress-induced TCF/LEF activity in MC3T3-E1 osteoblast cells using live cell imaging. We employed fluorescence resonance energy transfer (FRET)-based and green fluorescent protein (GFP)-based biosensors, which allowed us to monitor signal transduction in living cells in real time. Oscillatory (1Hz) shear stress (10 dynes/cm2) increased TCF/LEF activity and stimulated translocation of β-catenin to the nucleus with the distinct activity patterns of Rac1 and Cdc42. The shear stress-induced TCF/LEF activity was blocked by the inhibition of Rac1 and Cdc42 with their dominant negative mutants or selective drugs, but not by a dominant negative mutant of RhoA. In contrast, constitutively active Rac1 and Cdc42 mutants caused a significant enhancement of TCF/LEF activity. Moreover, activation of Rac1 and Cdc42 increased the basal level of TCF/LEF activity, while their inhibition decreased the basal level. Interestingly, disruption of cytoskeletal structures or inhibition of myosin activity did not significantly affect shear stress-induced TCF/LEF activity. Although Rac1 is reported to be involved in β-catenin in cancer cells, the involvement of Cdc42 in β-catenin signaling in osteoblasts has not been identified. Our findings in this study demonstrate that both Rac1 and Cdc42 GTPases are critical regulators in shear stress-driven β-catenin signaling in osteoblasts.
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    Synthesis and characterization of CaO-loaded electrospun matrices for bone tissue engineering
    (Springer, 2016-11) Münchow, Eliseu A.; Pankajakshan, Divya; Albuquerque, Maria T. P.; Kamocki, Krzysztof; Piva, Evandro; Gregory, Richard L.; Bottino, Marco C.; Biomedical and Applied Sciences, School of Dentistry
    Objectives To synthesize and characterize biodegradable polymer-based matrices loaded with CaO-nanoparticles for osteomyelitis treatment and bone tissue engineering. Materials and methods Poly(ε-caprolactone) (PCL) and PCL/gelatin (1:1, w/w) solutions containing CaO nanoparticles were electrospun into fibrous matrices. Scanning (SEM) and transmission (TEM) electron microscopy, Fourier Transformed Infrared (FTIR), Energy Dispersive X-ray Spectroscopy (EDS), contact angle (CA), tensile testing, and antibacterial activity (agar diffusion assay) against Staphylococcus aureus (S. aureus) were performed. Osteoprecursor cell (MC3T3-E1) response (i.e., viability and alkaline phosphatase expression/ALP) and infiltration into the matrices were evaluated. Results CaO nanoparticles were successfully incorporated into the fibers, with the median fiber diameter decreasing after CaO incorporation. The CA decreased with the 0addition of CaO, and the presence of gelatin made the matrix very hydrophilic (CA = 0°). Increasing CaO concentrations progressively reduced the mechanical properties (p≤0.030). CaO-loaded matrices did not display consistent antibacterial activity. MC3T3-E1 cell viability demonstrated the highest levels for CaO-loaded matrices containing gelatin after 7 days in culture. An increased ALP expression was consistently seen for PCL/CaO matrices when compared to PCL and gelatin-containing counterparts. Conclusions Despite inconsistent antibacterial activity, CaO nanoparticles can be effectively loaded into PCL or PCL/gelatin fibers without negatively affecting the overall performance of the matrices. More importantly, CaO incorporation enhanced cell viability as well as differentiation capacity, as demonstrated by an increased ALP expression. Clinical significance CaO-loaded electrospun matrices show potential for applications in bone tissue engineering.