Uniform tissues engineered by seeding and culturing cells in 3D scaffolds under perfusion at defined oxygen tensions

In this work, we assessed whether culture of uniformly seeded chondrocytes under direct perfusion, which supplies the cells with normoxic oxygen levels, can maintain a uniform distribution of viable cells throughout porous scaffolds several milimeters in thickness, and support the development of uniform tissue grafts. An integrated bioreactor system was first developed to streamline the steps of perfusion cell seeding of porous scaffolds and perfusion culture of the cell-seeded scaffolds. Oxygen tensions in perfused constructs were monitored by in-line oxygen sensors incorporated at the construct inlet and outlet. Adult human articular chondrocytes were perfusion-seeded into 4.5 mm thick foam scaffolds at a rate of 1 mm/s. Cell-seeded foams were then either cultured statically in dishes or further cultured under perfusion at a rate of 100 microm/s for 2 weeks. Following perfusion seeding, viable cells were uniformly distributed throughout the foams. Constructs subsequently cultured statically were highly heterogeneous, with cells and matrix concentrated at the construct periphery. In contrast, constructs cultured under perfusion were highly homogeneous, with uniform distributions of cells and matrix. Oxygen tensions of the perfused medium were maintained near normoxic levels (inlet congruent with 20%, outlet > 15%) at all times of culture. We have demonstrated that perfusion culture of cells seeded uniformly within porous scaffolds, at a flow rate maintaining a homogeneous oxygen supply, supports the development of uniform engineering tissue grafts of clinically relevant thicknesses.     

The Cassian patch reefs (lower Carnian,southern Alps)

The fauna of the upper Cassian Formation is composed mainly of reef-building and reef-dwelling organisms which occur as reeeposited material in basinal sediments, but have not been found as original reef bodies. Such bodies have now been discovered in the uppermost Cassian Formation of the central Dolomites from the Sella Group in the west to the Monti Cadini in the east. Generally they are small-scale patch reefs, not exceeding a few metres in thickness and lateral extent, which are intercalated in well-bedded detrital and micritic limestones. locally, larger biostromes spread out from the margins of the Cassian Dolomite buildups. Four types of faunal communities have been encountered in these reefs:

1. The thrombolite-calcareous algae community, composed of small patchy cryptalgal structures binding poorly sorted debris and associated with other Cyanophyta, sessile formainifera and scattered calcareous sponges and corals. This type is the most common within the calcareous and marly-tuffaceous facies of the Cassian Formation.
2. The calcareous sponge-coral community, composed mostly of calcareous sponges (stromatoporoids, some pharetronids) and, to a lesser extent, colonial corals and thrombolites. This community corre-sponds well to the Cassian reef fauna, best known from erratic blocks at Alpe di Specie, but has been found in situ only at one locality.
3. The Spongiomorpha-Solenopora community, associated with scattered calcareous sponges and colonial corals, forming a thin biostrome at one locality.
4. Coral communities, composed predominantly of colonial Scleractinia; found only in small or stratigraphically illdefined outcrops and in erratic blocks.

The Cassian patch reefs and biostromes mark the end of a basinal evolution which began in the Lower Ladinian, and the onset of newly expanding carbonate buildups of Cassian Dolomite. These buildups and the sponge-coral patch reefs might have been the source for the allochthonous reef fauna of the Cassian Formation which interfingers with both shallow water environments.     

Microbiome dynamics of human epidermis following skin barrier disruption

Background

Recent advances in sequencing technologies have enabled metagenomic analyses of many human body sites. Several studies have catalogued the composition of bacterial communities of the surface of human skin, mostly under static conditions in healthy volunteers. Skin injury will disturb the cutaneous homeostasis of the host tissue and its commensal microbiota, but the dynamics of this process have not been studied before. Here we analyzed the microbiota of the surface layer and the deeper layers of the stratum corneum of normal skin, and we investigated the dynamics of recolonization of skin microbiota following skin barrier disruption by tape stripping as a model of superficial injury.

Results

We observed gender differences in microbiota composition and showed that bacteria are not uniformly distributed in the stratum corneum. Phylogenetic distance analysis was employed to follow microbiota development during recolonization of injured skin. Surprisingly, the developing neo-microbiome at day 14 was more similar to that of the deeper stratum corneum layers than to the initial surface microbiome. In addition, we also observed variation in the host response towards superficial injury as assessed by the induction of antimicrobial protein expression in epidermal keratinocytes.

Conclusions

We suggest that the microbiome of the deeper layers, rather than that of the superficial skin layer, may be regarded as the host indigenous microbiome. Characterization of the skin microbiome under dynamic conditions, and the ensuing response of the microbial community and host tissue, will shed further light on the complex interaction between resident bacteria and epidermis.     

Computational evaluation of oxygen and shear stress distributions in 3D perfusion culture systems: macro-scale and micro-structured models

We present a combined macro-scale/micro-scale computational approach to quantify oxygen transport and flow-mediated shear stress to human chondrocytes cultured in three-dimensional scaffolds in a perfusion bioreactor system. A macro-scale model was developed to assess the influence of the bioreactor design and to identify the proper boundary conditions for the micro-scale model. The micro-scale model based on a micro-computed tomography (microCT) reconstruction of a poly(ethylene glycol terephthalate)/poly(butylene terephthalate) (PEGT/PBT) foam scaffold, was developed to assess the influence of the scaffold micro-architecture on local shear stress and oxygen levels within the scaffold pores. Experiments were performed to derive specific oxygen consumption rates for constructs perfused under flow rates of 0.3 and 0.03 ml min(-1). While macro-scale and micro-scale models predicted similar average oxygen levels at different depths within the scaffold, microCT models revealed small local oxygen variations within the scaffold micro-architecture. The combined macro-scale/micro-scale approach indicated that 0.3 ml min(-1), which subjected 95% of the cells to less than 6.3 mPa shear, would maintain the oxygen supply throughout the scaffold above anoxic levels (>1%), with 99.5% of the scaffold supplied with 8-2% O(2). Alternatively, at 0.03 ml min(-1), the macro-scale model predicted 6% of the cells would be supplied with 0.5-1% O(2), although this region of cells was confined to the periphery of the scaffold. Together with local variations predicted by the micro-scale model, the simulations underline that in the current model system, reducing the flow below 0.03 ml min(-1) would likely have dire consequences on cell viability to pronounced regions within the engineered construct. The presented approach provides a sensitive tool to aid efficient bioreactor optimization and scaffold design.     

Thermodynamic characterization of allosteric glycogen phosphorylase inhibitors

Glycogen phosphorylase (GP) is a validated target for the treatment of type 2 diabetes. Here we describe highly potent GP inhibitors, AVE5688, AVE2865, and AVE9423. The first two compounds are optimized members of the acyl urea series. The latter represents a novel quinolone class of GP inhibitors, which is introduced in this study. In the enzyme assay, both inhibitor types compete with the physiological activator AMP and act synergistically with glucose. Isothermal titration calorimetry (ITC) shows that the compounds strongly bind to nonphosphorylated, inactive GP (GPb). Binding to phosphorylated, active GP (GPa) is substantially weaker, and the thermodynamic profile reflects a coupled transition to the inactive (tense) conformation. Crystal structures confirm that the three inhibitors bind to the AMP site of tense state GP. These data provide the first direct evidence that acyl urea and quinolone compounds are allosteric inhibitors that selectively bind to and stabilize the inactive conformation of the enzyme. Furthermore, ITC reveals markedly different thermodynamic contributions to inhibitor potency that can be related to the binding modes observed in the cocrystal structures. For AVE5688, which occupies only the lower part of the bifurcated AMP site, binding to GPb (Kd = 170 nM) is exclusively enthalpic (Delta H = -9.0 kcal/mol, TDelta S = 0.3 kcal/mol). The inhibitors AVE2865 (Kd = 9 nM, Delta H = -6.8 kcal/mol, TDelta S = 4.2 kcal/mol) and AVE9423 (Kd = 24 nM, Delta H = -5.9 kcal/mol, TDelta S = 4.6 kcal/mol) fully exploit the volume of the binding pocket. Their pronounced binding entropy can be attributed to the extensive displacement of solvent molecules as well as to ionic interactions with the phosphate recognition site.     

Genetic paternity analyses in Little Owls (Athene noctua): does the high rate of paternal care select against extra-pair young?

Summary Previous studies have shown that extra-pair fertilizations are much less frequent in Non-Passeriformes, especially in raptors, than in Passeriformes. Low breeding densities, high breeding synchrony and high rates of paternal effort have been discussed as possible causes of these low extra-pair fertilization rates. Using DNA fingerprinting, we studied the mating system of Little Owls (Athene noctua) in a population of relatively high breeding density and comparatively low breeding synchrony. We found no cases of extra-pair fertilization among 53 nestlings of 16 breeding pairs. We conclude that paternal effort is probably the most important factor in preventing extra-pair fertilizations in Little Owls.

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Genetische Vaterschaftsanalysen bei Steinkäuzen (Athene noctua): Beeinflußt der hohe elterliche Aufwand der Männchen das Auftreten von Vaterschaften außerhalb des Paarbundes?

Zusammenfassung Zahlreiche Untersuchungen haben gezeigt, dass Befruchtungen außerhalb des Paarbundes bei Nicht-Singvogelarten wesentlich seltener vorkommen als bei Singvögeln. Dies gilt insbesondere auch für Greifvögel. Als Ursache für das seltene Auftreten von Befruchtungen außerhalb des Paarbundes in dieser Gruppe werden niedrige Brutpaardichten, eine hohe Brutsynchronisation und ein hoher elterlicher Aufwand auf Seiten der Männchen diskutiert. In der vorliegenden Studie haben wir das Paarungssystem des Steinkauzes (Athene noctua) in einer Population im Kreis Viersen (Niederrhein) mit Hilfe des DNA-Fingerprinting untersucht. Diese Population wies eine relativ hohe Brutpaardichte und eine vergleichsweise niedrige Brutsynchronisation auf. Bei der Analyse von 16 Bruten, die insgesamt 53 Nestlinge enthielten, konnte kein einziger Fall einer Befruchtung außerhalb des Paarbundes nachgewiesen werden. Dies führt uns zu dem Schluss, dass der wichtigste Faktor für die genetische Monogamie — zumindest beim Steinkauz — das hohe Maß des väterlichen Aufwandes bei der Brutversorgung ist.

     

Expression and localization of P-glycoprotein in human heart: effects of cardiomyopathy.

ABC-type transport proteins, such as P-glycoprotein (P-gp), modify intracellular concentrations of many substrate compounds. They serve as functional barriers against entry of xenobiotics (e.g., in the gut or the blood-brain barrier) or contribute to drug excretion. Expression of transport proteins in the heart could be an important factor modifying cardiac concentrations of drugs known to be transported by P-gp (e.g., beta-blockers, cardiac glycosides, doxorubicin). We therefore investigated the expression and localization of P-gp in human heart. Samples from 15 human hearts (left ventricle; five non-failing, five dilated cardiomyopathy, and five ischemic cardiomyopathy) were analyzed for expression of P-gp using real-time RT-PCR, immunohistochemistry, and in situ hybridization. Immunohistochemistry revealed expression of P-gp in endothelium of both arterioles and capillaries of all heart samples. Although P-gp mRNA was detected in all samples, its expression level was significantly reduced in patients with dilated cardiomyopathy. We describe variable expression of P-gp in human heart and its localization in the endothelial wall. Thus, intracardiac concentrations of various compounds may be modified, depending on the individual P-gp level.