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121.
IntroductionThe aim of this study was to investigate the effects of thyroid hormones tri-iodothyronine (T3), thyroxine (T4), and parathyroid hormone (PTH) from the parathyroid glands, known to regulate the developing limb and growth plate, on articular cartilage tissue regeneration using a scaffold-free in vitro model.MethodsIn Phase 1, T3, T4, or PTH was applied during weeks 1 or 3 of a 4-week neocartilage culture. Phase 2 employed T3 during week 1, followed by PTH during week 2, 3, or weeks 2 to 4, to further enhance tissue properties. Resultant neotissues were evaluated biochemically, mechanically, and histologically.ResultsIn Phase 1, T3 and T4 treatment during week 1 resulted in significantly enhanced collagen production; 1.4- and 1.3-times untreated neocartilage. Compressive and tensile properties were also significantly increased, as compared to untreated and PTH groups. PTH treatment did not result in notable tissue changes. As T3 induces hypertrophy, in Phase 2, PTH (known to suppress hypertrophy) was applied sequentially after T3. Excitingly, sequential treatment with T3 and PTH reduced expression of hypertrophic marker collagen X, while yielding neocartilage with significantly enhanced functional properties. Specifically, in comparison to no hormone application, these hormones increased compressive and tensile moduli 4.0-fold and 3.1-fold, respectively.ConclusionsThis study demonstrated that T3, together with PTH, when applied in a scaffold-free model of cartilage formation, significantly enhanced functional properties. The novel use of these thyroid hormones generates mechanically robust neocartilage via the use of a scaffold-free tissue engineering model.

Electronic supplementary material

The online version of this article (doi:10.1186/s13075-015-0541-5) contains supplementary material, which is available to authorized users.  相似文献   
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The Cu(II) center at the active site of the blue copper protein pseudoazurin from Alcaligenes faecalis has been substituted by Co(II) via denaturing of the protein, chelation and removal of copper by EDTA and refolding of the apo‐protein, followed by addition of an aqueous solution of CoCl2. Sitting drop vapour diffusion experiments produced green hexagonal crystals, which belong to space group P65, with unit cell dimensions a = b = 50.03, c = 98.80 Å. Diffraction data, collected at 291 K on a copper rotating anode X‐ray source, were phased by the anomalous signal of the cobalt atom. The structure was built automatically, fitted manually and subsequently refined to 1.86 Å resolution. The Co‐substituted protein exhibits similar overall geometry to the native structure with copper. Cobalt binds more strongly to the axial Met86‐Sδ and retains the tetrahedral arrangement with the four ligand atoms, His40‐Nδ1, Cys78‐Sγ, His81‐Nδ1, and 86Met‐Sδ, although the structure is less distorted than the native copper protein. The structure reported herein, is the first crystallographic structure of a Co(II)‐substituted pseudoazurin. © 2010 Wiley Periodicals, Inc. Biopolymers 95: 202–207, 2011.  相似文献   
124.
The dynamics of isogenic cell populations can be described by cell population balance models that account for phenotypic heterogeneity. To utilize the predictive power of these models, however, we must know the rates of single-cell reaction and division and the bivariate partition probability density function. These three intrinsic physiological state (IPS) functions can be obtained by solving an inverse problem that requires knowledge of the phenotypic distributions for the overall cell population, the dividing cell subpopulation and the newborn cell subpopulation. We present here a robust computational procedure that can accurately estimate the IPS functions for heterogeneous cell populations. A detailed parametric analysis shows how the accuracy of the inverse solution is affected by discretization parameters, the type of non-parametric estimators used, the qualitative characteristics of phenotypic distributions and the unknown partitioning probability density function. The effect of finite sampling and measurement errors on the accuracy of the recovered IPS functions is also assessed. Finally, we apply the procedure to estimate the IPS functions of an E. coli population carrying an IPTG-inducible genetic toggle network. This study completes the development of an integrated experimental and computational framework that can become a powerful tool for quantifying single-cell behavior using measurements from heterogeneous cell populations.  相似文献   
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Central to understanding mechanotransduction in the knee meniscus is the characterization of meniscus cell mechanics. In addition to biochemical and geometric differences, the inner and outer regions of the meniscus contain cells that are distinct in morphology and phenotype. This study investigated the regional variation in meniscus cell mechanics in comparison with articular chondrocytes and ligament cells. It was found that the meniscus contains two biomechanically distinct cell populations, with outer meniscus cells being stiffer (1.59 ± 0.19 kPa) than inner meniscus cells (1.07 ± 0.14 kPa). Additionally, it was found that both outer and inner meniscus cell stiffnesses were similar to ligament cells (1.32 ± 0.20 kPa), and articular chondrocytes showed the highest stiffness overall (2.51 ± 0.20 kPa). Comparison of compressibility characteristics of the cells showed similarities between articular chondrocytes and inner meniscus cells, as well as between outer meniscus cells and ligament cells. These results show that cellular biomechanics vary regionally in the knee meniscus and that meniscus cells are biomechanically similar to ligament cells. The mechanical properties of musculoskeletal cells determined in this study may be useful for the development of mathematical models or the design of experiments studying mechanotransduction in a variety of soft tissues.  相似文献   
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Pseudotyping viral vectors with vesicular stomatitis virus glycoprotein (VSV-G) enables the transduction of an extensive range of cell types from different species. We have discovered two important parameters of the VSV-G-pseudotyping phenomenon that relate directly to the transduction potential of lentiviral vectors: (1) the glycosylation status of VSV-G, and (2) the quantity of glycoprotein associated with virions. We measured production-cell and virion-associated quantities of two isoform variants of VSV-G, which differ in their glycosylation status, VSV-G1 and VSV-G2, and assessed the impact of this difference on the efficiency of mammalian cell transduction by lentiviral vectors. The glycosylation of VSV-G at N336 allowed greater maximal expression of VSV-G in HEK293T cells, thus facilitating vector pseudotyping. The transduction of primate cell lines was substantially affected (up to 50-fold) by the degree of VSV-G1 or VSV-G2 incorporation, whereas other cell lines, such as D17 (canine), were less sensitive to virion-associated VSV-G1/2 quantities. These data indicate that the minimum required concentration of virion-associated VSV-G differs substantially between cell species/types. The implications of these data with regard to VSV-G-pseudotyped vector production, titration, and use in host-cell restriction studies, are discussed.  相似文献   
128.
The cytoskeleton is known to play an important role in the biomechanical nature and structure of cells, but its particular function in compressive characteristics has not yet been fully examined. This study focused on the contribution of the main three cytoskeletal elements to the bulk compressive stiffness (as measured by the compressive modulus), volumetric or apparent compressibility changes (as further indicated by apparent Poisson's ratio), and recovery behavior of individual chondrocytes. Before mechanical testing, cytochalasin D, acrylamide, or colchicine was used to disrupt actin microfilaments, intermediate filaments, or microtubules, respectively. Cells were subjected to a range of compressive strains and allowed to recover to equilibrium. Analysis of the video recording for each mechanical event yielded relevant compressive properties and recovery characteristics related to the specific cytoskeletal disrupting agent and as a function of applied axial strain. Inhibition of actin microfilaments had the greatest effect on bulk compressive stiffness (∼50% decrease compared to control). Meanwhile, intermediate filaments and microtubules were each found to play an integral role in either the diminution (compressibility) or retention (incompressibility) of original cell volume during compression. In addition, microtubule disruption had the largest effect on the “critical strain threshold” in cellular mechanical behavior (33% decrease compared to control), as well as the characteristic time for recovery (∼100% increase compared to control). Elucidating the role of the cytoskeleton in the compressive biomechanical behavior of single cells is an important step toward understanding the basis of mechanotransduction and the etiology of cellular disease processes.  相似文献   
129.
The way in which the nucleus experiences mechanical forces has important implications for understanding mechanotransduction. Knowledge of nuclear material properties and, specifically, their relationship to the properties of the bulk cell can help determine if the nucleus directly experiences mechanical loads, or if it is a signal transduction mechanism secondary to cell membrane deformation that leads to altered gene expression. Prior work measuring nuclear material properties using micropipette aspiration suggests that the nucleus is substantially stiffer than the bulk cell [Guilak, F., Tedrow, J.R., Burgkart, R., 2000. Viscoelastic properties of the cell nucleus. Biochem. Biophys. Res. Commun. 269, 781–786], whereas recent work with unconfined compression of single chondrocytes showed a nearly one-to-one correlation between cellular and nuclear strains [Leipzig, N.D., Athanasiou, K.A., 2008. Static compression of single chondrocytes catabolically modifies single-cell gene expression. Biophys. J. 94, 2412–2422]. In this study, a linearly elastic finite element model of the cell with a nuclear inclusion was used to simulate the unconfined compression data. Cytoplasmic and nuclear stiffnesses were varied from 1 to 7 kPa for several combinations of cytoplasmic and nuclear Poisson's ratios. It was found that the experimental data were best fit when the ratio of cytoplasmic to nuclear stiffness was 1.4, and both cytoplasm and nucleus were modeled as incompressible. The cytoplasmic to nuclear stiffness ratio is significantly lower than prior reports for isolated nuclei. These results suggest that the nucleus may behave mechanically different in situ than when isolated.  相似文献   
130.
The mutagenic and clastogenic effects of the antineoplastic agents homo-aza-steroidal ester (ASE) and chlorambucil (CBC) were tested for their ability to induce mutations in the Salmonella/microsome system and SCE in CHO cells in culture. ASE was found to be positive in strains TA1535 and TA100 and in the newer strain TA102 with and without metabolic activation, while CBC caused histidine reversion in strain TA102 after the addition of mammalian liver microsomal extract (S9). In addition, both agents were found to be strongly positive for SCE induction. The mutagenic and clastogenic actions of both agents were of a dose-response type.  相似文献   
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