Passive and active contributions to generated force and retraction in heart valve tissue engineering

In tissue engineered heart valves, cell-mediated stress development during culture results in leaflet retraction at time of implantation. This tissue retraction is partly active due to traction forces exerted by the cells and partly passive due to release of residual stress in the extracellular matrix and the cells. Within this study, we unraveled the passive and active contributions of cells and matrix to generated force and retraction in engineered heart valve tissues. Tissue engineered rectangular strips, fabricated from PGA/P4HB scaffolds and seeded with human myofibroblasts, were cultured for 4 weeks, after which the cellular contribution was changed at different levels. Elimination of the active cellular traction forces was achieved with Cytochalasin D and inhibition of the Rho-associated kinase pathway. Both active and passive cellular contributions were eliminated by lysation and/or decellularization of the tissue. Maximum cell activity was reached by increasing the fetal bovine serum concentration to 50%. The generated force decreased ~20% after elimination of the active cellular component, ~25% when the passive cellular component was removed as well and remained unaffected by increased serum concentrations. Passive retraction accounted for ~60% of total retraction, of which ~15% was residual stress in the matrix and ~45% was passive cell retraction. Cell traction forces accounted for the remainder ~40% of the retraction. Full activation of the cells increased retraction by ~45%. These results illustrate the importance of the cells in the process of tissue retraction, not only actively retracting the tissue, but also in a passive manner to a large extent.     

A Finite Element Approach for Skeletal Muscle using a Distributed Moment Model of Contraction

The present paper describes a geometrically and physically nonlinear continuum model to study the mechanical behaviour of passive and active skeletal muscle. The contraction is described with a Huxley type model. A Distributed Moments approach is used to convert the Huxley partial differential equation in a set of ordinary differential equations. An isoparametric brick element is developed to solve the field equations numerically. Special arrangements are made to deal with the combination of highly nonlinear effects and the nearly incompressible behaviour of the muscle. For this a Natural Penalty Method (NPM) and an Enhanced Stiffness Method (ESM) are tested. Finally an example of an analysis of a contracting tibialis anterior muscle of a rat is given. The DM-method proved to be an efficient tool in the numerical solution process. The ESM showed the best performance in describing the incompressible behaviour.     

Passive transverse mechanical properties as a function of temperature of rat skeletal muscle in vitro

The objective of the current study was to determine the in vitro passive transverse mechanical properties of skeletal muscle with Dynamic Mechanical Thermal Analysis (DMTA) tests. The starting hypotheses was that the time-temperature-superposition principle could be used to expand the DMTA results to a 1 kHz frequency range. Experiments were performed with rat hind leg skeletal muscle tissue samples on a rotational rheometer using a parallel plate geometry. Because of the small size and low modulus of the samples, the standard test geometry was altered and the samples were shifted from the center to the edge of the plates. From strain sweep tests it became clear that for strains smaller than 0.003 the muscle tissue behaves linearly. In the linear region storage moduli ranged between 24 kPa (omega = 1 rad/s) and 42 kPa (omega = 100 rad/s) at T = 4 degrees C and 22 kPa and 33 kPa at 29 degrees C within the experimental frequency range. The loss modulus decreased with increasing frequency and ranged between 7 and 4 kPa at 4 degrees C and 4.5 and 3.5 kPa at 29 degrees C. Although the properties are clearly temperature dependent, a temperature shift in phase angle delta could not be detected, thus Time Temperature Superposition is not allowed for skeletal muscle in vitro.     

Characterisation of Anisotropic and Non-linear Behaviour of Human Skin In Vivo

The aim of this work is to characterise the in-plane mechanical behaviour of human skin in vivo. For this purpose the structural skin model proposed by Lanir [1] is employed and a mixed numerical-experimental method is developed. The numerical-experimental method is based on the confrontation of measured data from an experiment, with calculated data from a finite element model, eventually leading to the determination of some of the parameters of a constitutive model, in the present case Lanir's Skin Model. Since collagen, the main constituent of skin, dominates the anisotropic and non-linear behaviour of skin, the parameters of Lanir's Skin Model concerning the mechanical behaviour of the collagen fibres are estimated. In vivo experiments were carried out on the volar forearm. During the experiments, reaction forces and the displacement field at different states of deformation are measured. Both data sets are used for the determination of the parameters.     

Frequency doubling in the cyanobacterial circadian clock

Organisms use circadian clocks to generate 24‐h rhythms in gene expression. However, the clock can interact with other pathways to generate shorter period oscillations. It remains unclear how these different frequencies are generated. Here, we examine this problem by studying the coupling of the clock to the alternative sigma factor sigC in the cyanobacterium Synechococcus elongatus. Using single‐cell microscopy, we find that psbAI, a key photosynthesis gene regulated by both sigC and the clock, is activated with two peaks of gene expression every circadian cycle under constant low light. This two‐peak oscillation is dependent on sigC, without which psbAI rhythms revert to one oscillatory peak per day. We also observe two circadian peaks of elongation rate, which are dependent on sigC, suggesting a role for the frequency doubling in modulating growth. We propose that the two‐peak rhythm in psbAI expression is generated by an incoherent feedforward loop between the clock, sigC and psbAI. Modelling and experiments suggest that this could be a general network motif to allow frequency doubling of outputs.     

Can three incongruence tests predict when data should be combined?

Advocates of conditional combination have argued that testing for incongruence between data partitions is an important step in data exploration. Unless the partitions have had distinct histories, as in horizontal gene transfer, incongruence means that one or more data support the wrong phylogeny. This study examines the relationship between incongruence and phylogenetic accuracy using three tests of incongruence. These tests were applied to pairs of mitochondrial DNA data partitions from two well-corroborated vertebrate phylogenies. Of the three tests, the most useful was the incongruence length difference test (ILD, also called the partition homogeneity test). This test distinguished between cases in which combining the data generally improved phylogenetic accuracy (P > 0.01) and cases in which accuracy of the combined data suffered relative to the individual partitions (P < 0.001). In contrast, in several cases, the Templeton and Rodrigo tests detected highly significant incongruence (P < 0.001) even though combining the incongruent partitions actually increased phylogenetic accuracy. All three tests identified cases in which improving the reconstruction model would improve the phylogenetic accuracy of the individual partitions.      

Complete nucleotide sequences of bovine alpha S2- and beta-casein cDNAs: comparisons with related sequences in other species

The nucleotide sequences corresponding to bovine alpha S2- and beta- casein mRNAs have been determined by cDNA analysis. Both sequences appear to be complete at their 5' ends. The nucleotide sequence of alpha S2-casein, when compared with the corresponding cavine A sequence, helps to define the boundaries of a large amino acid repeat (approximately 80 residues) whereas comparisons with the nucleotide sequences of rat gamma- and mouse epsilon-casein mRNAs also reveal extensive sequence similarities. An alignment of these four sequences shows that the divergence of their translated regions has been characterized by the duplication and deletion of discrete segments of sequence that probably correspond to exons. A high degree of nucleotide substitution is also found when the four sequences are compared, except for well-conserved leader-peptide and phosphorylation-site sequences and, to a lesser extent, the 5'-untranslated regions. Similar comparison of the bovine and rat beta-caseins shows that their divergence has involved a high rate of nucleotide substitution but that no major insertions or deletions of sequence have occurred. The several splice sites that have veen defined in the rat beta-casein gene are likely to have been conserved in the bovine. The contrasting evolutionary histories of the alpha- and beta-casein coding sequences correlate with the distinctive functions of these proteins in the casein micelle system in milk.