Mineralization of calcium phosphate crystals in starch template inducing a brushite kidney stone biomimetic composite

Dicalcium phosphate dihydrate (brushite) and octacalcium phosphate (OCP) crystals are precursors of hydroxyapatite (HAp) for tooth enamel, dentine, and bones formation in living organisms. Here, we introduce a new method for biomimicking brushite and OCP in starch using single and double diffusion techniques. Brushite and OCP crystals were grown by precipitation in starch after gelation. The obtained materials were analyzed by infrared spectroscopy (IR), scanning electron microscopy (SEM), X-ray diffraction (XRD), and confocal laser scanning microscopy (CLSM). IR spectra demonstrate starch inclusion by peak shifts in the 2900–3500 cm–1 region. SEM showed two different morphologies: plate-shaped and needle-like crystals. Calcium phosphate/starch aggregates bear strong resemblance to prismatic brushite kidney stones. This may open up a clue to understand the mechanism of kidney stone formation.

Mesenchymal stem cells and collagen patches for anterior cruciate ligament repair

AIM: To investigate collagen patches seeded with mesenchymal stem cells (MSCs) and/or tenocytes (TCs) with regards to their suitability for anterior cruciate ligament (ACL) repair. METHODS: Dynamic Intraligamentary Stabilization (DIS) utilizes a dynamic screw system to keep ACL remnants in place and promote biological healing, supplemented by collagen patches. How these scaffolds interact with cells and what type of benefit they provide has not yet been investigated in detail. Primary ACL-derived TCs and human bone marrow derived MSCs were seeded onto two different types of 3D collagen scaffolds, Chondro-Gide® (CG) and Novocart® (NC). Cells were seeded onto the scaffolds and cultured for 7 days either as a pure populations or as “premix” containing a 1 : 1 ratio of TCs to MSCs. Additionally, as controls, cells were seeded in monolayers and in co-cultures on both sides of porous high-density membrane inserts (0.4µm). We analyzed the patches by real time polymerase chain reaction (RT-PCR), glycosaminoglycan (GAG), DNA and hydroxy-proline (HYP) content, was determined. To determine cell spreading and adherence in the scaffolds microscopic imaging techniques, i.e. confocal laser scanning microscopy (cLSM) and scanning electron microscopy (SEM), were applied. RESULTS: CLSM and SEM imaging analysis confirmed cell adherence onto scaffolds. The metabolic cell activity revealed that patches promote adherence and proliferation of cells. The most dramatic increase in absolute metabolic cell activity was measured for CG samples seeded with tenocytes or a 1:1 cell premix. Analysis of DNA content and cLSM imaging also indicated MSCs were not proliferating as nicely as tenocytes on CG. The HYP to GAG ratio significantly changed for the premix group, resulting from a slightly lower GAG content, demonstrating that the cells are modifying the underlying matrix. Real-time quantitative polymerase chain reaction data indicated that MSCs showed a trend of differentiation towards a more tenogenic-like phenotype after 7 days. CONCLUSION: CG and NC are both cyto-compatible with primary MSCs and TCs; TCs seemed to perform better on these collagen patches than MSCs.

Fluorescence-encoded gold nanoparticles: library design and modulation of cellular uptake into dendritic cells.

In order to harness the unique properties of nanoparticles for novel clinical applications and to modulate their uptake into specific immune cells we designed a new library of homo- and hetero-functional fluorescence-encoded gold nanoparticles (Au-NPs) using different poly(vinyl alcohol) and poly(ethylene glycol)-based polymers for particle coating and stabilization. The encoded particles were fully characterized by UV-Vis and fluorescence spectroscopy, zeta potential and dynamic light scattering. The uptake by human monocyte derived dendritic cells in vitro was studied by confocal laser scanning microscopy and quantified by fluorescence-activated cell sorting and inductively coupled plasma atomic emission spectroscopy. We show how the chemical modification of particle surfaces, for instance by attaching fluorescent dyes, can conceal fundamental particle properties and modulate cellular uptake. In order to mask the influence of fluorescent dyes on cellular uptake while still exploiting its fluorescence for detection, we have created hetero-functionalized Au-NPs, which again show typical particle dependent cellular interactions. Our study clearly prove that the thorough characterization of nanoparticles at each modification step in the engineering process is absolutely essential and that it can be necessary to make substantial adjustments of the particles in order to obtain reliable cellular uptake data, which truly reflects particle properties.

Autonomic angiotensinergic fibres in the human heart with an efferent sympathetic cophenotype

AIM The autonomic innervation of the heart consists of sympathetic and parasympathetic nerve fibres, and fibres of the intrinsic ganglionated plexus with noradrenaline and acytylcholine as principal neurotransmitters. The fibres co-release neuropeptides to modulate intracardiac neurotransmission by specific presynaptic and postsynaptic receptors. The coexpression of angiotensin II in sympathetic fibres of the human heart and its role are not known so far. METHODS Autopsy specimens of human hearts were studied (n=3; ventricles). Using immunocytological methods, cryostat sections were stained by a murine monoclonal antibody (4B3) directed against angiotensin II and co-stained by polyclonal antibodies against tyrosine hydroxylase, a catecholaminergic marker. Visualisation of the antibodies was by confocal light microscopy or laser scanning microscopy. RESULTS Angiotensin II-positive autonomic fibres with and without a catecholaminergic cophenotype (hydroxylase-positive) were found in all parts of the human ventricles. In the epicardium, the fibres were grouped in larger bundles of up to 100 and more fibres. They followed the preformed anatomic septa and epicardial vessels towards the myocardium and endocardium where the bundles dissolved and the individual fibres spread between myocytes and within the endocardium. Generally, angiotensinergic fibres showed no synaptic enlargements or only a few if they were also catecholaminergic. The exclusively catechalominergic fibres were characterised by multiple beaded synapses. CONCLUSION The autonomic innervation of the human heart contains angiotensinergic fibres with a sympathetic efferent phenotype and exclusively angiotensinergic fibers representing probably afferents. Angiotensinergic neurotransmission may modulate intracardiac sympathetic and parasympathetic activity and thereby influence cardiac and circulatory function.

Establishment of a confluent monolayer model with human primary trophoblast cells: novel insights into placental glucose transport

STUDY HYPOTHESIS Using optimized conditions, primary trophoblast cells isolated from human term placenta can develop a confluent monolayer in vitro, which morphologically and functionally resembles the microvilli structure found in vivo. STUDY FINDING We report the successful establishment of a confluent human primary trophoblast monolayer using pre-coated polycarbonate inserts, where the integrity and functionality was validated by cell morphology, biophysical features, cellular marker expression and secretion, and asymmetric glucose transport. WHAT IS KNOWN ALREADY Human trophoblast cells form the initial barrier between maternal and fetal blood to regulate materno-fetal exchange processes. Although the method for isolating pure human cytotrophoblast cells was developed almost 30 years ago, a functional in vitro model with primary trophoblasts forming a confluent monolayer is still lacking. STUDY DESIGN, SAMPLES/MATERIALS, METHODS Human term cytotrophoblasts were isolated by enzymatic digestion and density gradient separation. The purity of the primary cells was evaluated by flow cytometry using the trophoblast-specific marker cytokeratin 7, and vimentin as an indicator for potentially contaminating cells. We screened different coating matrices for high cell viability to optimize the growth conditions for primary trophoblasts on polycarbonate inserts. During culture, cell confluency and polarity were monitored daily by determining transepithelial electrical resistance (TEER) and permeability properties of florescent dyes. The time course of syncytia-related gene expression and hCG secretion during syncytialization were assessed by quantitative RT-PCR and enzyme-linked immunosorbent assay, respectively. The morphology of cultured trophoblasts after 5 days was determined by light microscopy, scanning electron microscopy (SEM) and transmission electron microscopy (TEM). Membrane makers were visualized using confocal microscopy. Additionally, glucose transport studies were performed on the polarized trophoblasts in the same system. MAIN RESULTS AND THE ROLE OF CHANCE During 5-day culture, the highly pure trophoblasts were cultured on inserts coated with reconstituted basement membrane matrix . They exhibited a confluent polarized monolayer, with a modest TEER and a size-dependent apparent permeability coefficient (Papp) to fluorescently labeled compounds (MW ∼400-70 000 Da). The syncytialization progress was characterized by gradually increasing mRNA levels of fusogen genes and elevating hCG secretion. SEM analyses confirmed a confluent trophoblast layer with numerous microvilli, and TEM revealed a monolayer with tight junctions. Immunocytochemistry on the confluent trophoblasts showed positivity for the cell-cell adhesion molecule E-cadherin, the tight junction protein 1 (ZO-1) and the membrane proteins ATP-binding cassette transporter A1 (ABCA1) and glucose transporter 1 (GLUT1). Applying this model to study the bidirectional transport of a non-metabolizable glucose derivative indicated a carrier-mediated placental glucose transport mechanism with asymmetric kinetics. LIMITATIONS, REASONS FOR CAUTION The current study is only focused on primary trophoblast cells isolated from healthy placentas delivered at term. It remains to be evaluated whether this system can be extended to pathological trophoblasts isolated from diverse gestational diseases. WIDER IMPLICATIONS OF THE FINDINGS These findings confirmed the physiological properties of the newly developed human trophoblast barrier, which can be applied to study the exchange of endobiotics and xenobiotics between the maternal and fetal compartment, as well as intracellular metabolism, paracellular contributions and regulatory mechanisms influencing the vectorial transport of molecules. LARGE-SCALE DATA Not applicable. STUDY FUNDING AND COMPETING INTERESTS This study was supported by the Swiss National Center of Competence in Research, NCCR TransCure, University of Bern, Switzerland, and the Swiss National Science Foundation (grant no. 310030_149958, C.A.). All authors declare that their participation in the study did not involve factual or potential conflicts of interests.