Optimized procedures for testing plasma metanephrines in patients on hemodialysis.

Diagnosis of pheochromocytomas and paragangliomas in patients receiving hemodialysis is troublesome. The aim of the study was to establish optimal conditions for blood sampling for mass spectrometric measurements of normetanephrine, metanephrine and 3-methoxytyramine in patients on hemodialysis and specific reference intervals for plasma metanephrines under the most optimal sampling conditions. Blood was sampled before and near the end of dialysis, including different sampling sites in 170 patients on hemodialysis. Plasma normetanephrine concentrations were lower (P < 0.0001) and metanephrine concentrations higher (P < 0.0001) in shunt than in venous blood, with no differences for 3-methoxytyramine. Normetanephrine, metanephrine and 3-methoxytyramine concentrations in shunt and venous blood were lower (P < 0.0001) near the end than before hemodialysis. Upper cut-offs for normetanephrine were 34% lower when the blood was drawn from the shunt and near the end of hemodialysis compared to blood drawn before hemodialysis. This study establishes optimal sampling conditions using blood from the dialysis shunt near the end of hemodialysis with optimal reference intervals for plasma metanephrines for the diagnosis of pheochromocytomas/paragangliomas among patients on hemodialysis.

ROS Dependent Wnt/ß-Catenin Pathway and Its Regulation on Defined Micro-Pillars-A Combined In Vitro and In Silico Study.

The physico-chemical surface design of implants influences the surrounding cells. Osteoblasts on sharp-edged micro-topographies revealed an impaired cell phenotype, function and Ca2+ mobilization. The influence of edges and ridges on the Wnt/ß-catenin pathway in combination with the cells' stress response has not been clear. Therefore, MG-63 osteoblasts were studied on defined titanium-coated micro-pillars (5 × 5 × 5 µm) in vitro and in silico. MG-63s on micro-pillars indicated an activated state of the Wnt/ß-catenin pathway. The ß-catenin protein accumulated in the cytosol and translocated into the nucleus. Gene profiling indicated an antagonism mechanism of the transcriptional activity of ß-catenin due to an increased expression of inhibitors like ICAT (inhibitor of ß-catenin and transcription factor-4). Cells on pillars produced a significant reactive oxygen species (ROS) amount after 1 and 24 h. In silico analyses provided a detailed view on how transcriptional activity of Wnt signaling is coordinated in response to the oxidative stress induced by the micro-topography. Based on a coordinated expression of regulatory elements of the Wnt/ß-catenin pathway, MG-63s are able to cope with an increased accumulation of ß-catenin on micro-pillars and suppress an unintended target gene expression. Further, ß-catenin may be diverted into other signaling pathways to support defense mechanisms against ROS.

Enhanced calcium ion mobilization in osteoblasts on amino group containing plasma polymer nanolayer.

BACKGROUND: Biomaterial modifications-chemical and topographical-are of particular importance for the integration of materials in biosystems. Cells are known to sense these biomaterial characteristics, but it has remained unclear which physiological processes bio modifications trigger. Hence, the question arises of whether the dynamic of intracellular calcium ions is important for the characterization of the cell-material interaction. In our prior research we could demonstrate that a defined geometrical surface topography affects the cell physiology; this was finally detectable in a reduced intracellular calcium mobilization after the addition of adenosine triphosphate (ATP). RESULTS: This new contribution examines the cell physiology of human osteoblasts concerning the relative cell viability and the calcium ion dynamic on different chemical modifications of silicon-titanium (Ti) substrates. Chemical modifications comprising the coating of Ti surfaces with a plasma polymerized allylamine (PPAAm)-layer or with a thin layer of collagen type-I were compared with a bare Ti substrate as well as tissue culture plastic. For this purpose, the human osteoblasts (MG-63 and primary osteoblasts) were seeded onto the surfaces for 24 h. The relative cell viability was determined by colorimetric measurements of the cell metabolism and relativized to the density of cells quantified using crystal violet staining. The calcium ion dynamic of osteoblasts was evaluated by the calcium imaging analysis of fluo-3 stained vital cells using a confocal laser scanning microscope. The positively charged nano PPAAm-layer resulted in enhanced intracellular calcium ion mobilization after ATP-stimulus and cell viability. This study underlines the importance of the calcium signaling for the manifestation of the cell physiology. CONCLUSIONS: Our current work provides new insights into the intracellular calcium dynamic caused by diverse chemical surface compositions. The calcium ion dynamic appears to be a sensitive parameter for the cell physiology and, thus, may represent a useful approach for evaluating a new biomaterial. In this regard, reliable in vitro-tests of cell behavior at the interface to a material are crucial steps in securing the success of a new biomaterial in medicine.

Sensing of micropillars by osteoblasts involves complex intracellular signaling.

Topographical material surface features are sensed by cells and provoke a large range of cellular responses. We recognized earlier, that at micropillar topographies in the range of 5 µm, the osteoblasts attempt to phagocytize the pillars resulted in increased energy requirements and reduced osteoblast marker expression, e.g., collagen type I and osteocalcin. However, the precise cellular signaling transducing the topographic information into the cell and evoking phagocytic processes remained unknown. Here, we could show that the RhoA/ROCK signaling is involved in the transduction of the topography-mediated cellular reactions. After inhibition of ROCK-2 with Y27632 for 24 h, no caveolae-mediated micropillar assembly of the cell membrane domain component caveolin-1 (Cav-1) was found. ROCK inhibition was also able to attenuate the pillar-induced decrease in ß-actin. Interestingly, phosphatidylinositol 3-kinase (PI3K) inhibition with LY294002 for 24 h did not influence the Cav-1 clustering on micropillars. Our results illustrate the importance of the integrin down-stream signaling of RhoA/ROCK in the recognition of and adaption to surface microtopographies by osteoblasts and extend our understanding about the complex mechanism of action inside the cells.

Data supporting attempted caveolae-mediated phagocytosis of surface-fixed micro-pillars by human osteoblasts.

The provided data contains the phagocytic interaction of human MG-63 osteoblasts with micro-particles 6 µm in size as well as geometric micro-pillared topography with micro-pillar sizes 5 µm of length, width, height and spacing respectively related to the research article entitled "Attempted caveolae-mediated phagocytosis of surface-fixed micro-pillars by human osteoblasts" in the Biomaterials journal. [1] Micro-particle treatment was used as positive control triggering phagocytosis by the osteoblasts. Caveolin-1 (Cav-1) as major structural component of caveolae [2] plays an important role in the phagocytic process of micro-particles and -pillars. Data related to the experiments in [1] with siRNA-mediated knockdown are presented here as well as micro-particle control experiments, tubulin analysis on the micro-pillared topography and initial cell interaction with the micro-pillars.

Attempted caveolae-mediated phagocytosis of surface-fixed micro-pillars by human osteoblasts.

Cells are sensitive to their underlying micro- and nano-topography, but the complex interplay is not completely understood especially if sharp edges and ridges of stochastically modified surfaces interfere with an attached cell body. Micro-topography offers cues that evoke a large range of cell responses e.g. altered adhesion behavior and integrin expression resulting in disturbed cell functions. In this study, we analyzed why osteoblastic cells mimic the underlying geometrical micro-pillar structure (5 × 5 × 5 µm, spacing of 5 µm) with their actin cytoskeleton. Interestingly, we discovered an attempted caveolae-mediated phagocytosis of each micro-pillar beneath the cells, which was accompanied by increased intracellular reactive oxygen species (ROS) production and reduced intracellular ATP levels. This energy consuming process hampered the cells in their function as osteoblasts at the interface. The raft-dependent/caveolae-mediated phagocytic pathway is regulated by diverse cellular components including caveolin-1 (Cav-1), cholesterol, actin cytoskeleton as well as actin-binding proteins like annexin A2 (AnxA2). Our results show a new aspect of osteoblast-material interaction and give insight into how cells behave on extraordinary micro-structures. We conclude that stochastically structured implants used in orthopedic surgery should avoid any topographical heights which induce phagocytosis to prevent their successful ingrowth.

The potential role of human osteoblasts for periprosthetic osteolysis following exposure to wear particles.

Aseptic loosening in total hip replacement is mainly caused by wear particles inducing inflammation and osteolysis. Wear can be a consequence of micromotions at the interface between implant and bone cement. Due to complex cellular interactions, different mediators (e.g. cytokines, proteinases) are released, which can promote osteolytic processes in the periprosthetic tissue followed by loosening of the implant. Furthermore, a reduced matrix synthesis and an induced apoptosis rate can be observed. The purpose of this study was to evaluate to what extent human primary osteoblasts exposed to wear particles are involved in the osteolysis. The viability, the secretion of collagen and collagenases and the variety of released cytokines after particle exposure was examined. Therefore, human osteoblasts were incubated with particles experimentally generated in the interface between hip stems with rough and smooth surface finishings as well as different material compositions (Ti-6Al-7Nb, Co-28Cr-6Mo and 316L) and bone cement mantle made of Palacos R containing zirconium oxide particles. Commercially pure titanium particles, titanium oxide, polymethylmethacrylate and particulate zirconium oxide were used as references. The results revealed distinct effects on the cytokine release of human osteoblasts towards particulate debris. Thereby, human osteoblasts released increased levels of interleukine (IL)-6 and IL-8 after treatment with metallic wear particles. The expression of VEGF was slightly induced by all particle entities at lower concentrations. Apoptotic rates were enhanced for osteoblasts exposed to all the tested particles. Furthermore, the de novo synthesis of type 1 collagen was reduced and the expression of the matrix metalloproteinase (MMP)-1 was considerably increased. However, wear particles of Co-28Cr-6Mo stems seemed to be more aggressive, whereas particles derived from stainless steel stems caused less adverse cellular reaction. Among the reference particles, which caused less altered reactions in the metabolism of osteoblasts in general, ZrO2 can be assumed as the material with the smallest cell biological effects.

The effect of positively charged plasma polymerization on initial osteoblastic focal adhesion on titanium surfaces.

The crucial factor of metal implant ingrowth in the bone is the rapid cellular acceptance. Therefore, the knowledge about additionally used adhesion mechanisms of osteoblasts, like their negatively charged hyaluronan coat, generates new surface functionalization strategies. Here, titanium was coated with a very thin, adherent, cross-linked, pinhole- and additive-free allylamine plasma polymer layer (PPAAm) resistant to hydrolysis and delamination and equipped with a high density of positively charged amino groups. This plasma polymer-functionalization of titanium is advantageous concerning osteoblastic focal adhesion formation as vinculin and paxillin, actin cytoskeleton development and, in consequence in differentiated cell functions, compared to a pure titanium surface-but similar such as the collagen I bonded surface via a polyethylenglycol-diacid (PEG DA)-spacer.

Modalities of testing Helicobacter pylori in patients with nonmalignant bile duct diseases.

AIM: This paper describes the procedure of detection of Helicobacter pylori (H. pylori) in bile specimens in patients suffering from benign diseases of biliary ducts (lithiasis with/without nonspecific cholangitis). METHODS: The group of 72 patients entering the study consisted of 32 male and 40 female (45% and 55%, respectively). Bile was obtained during ERCP in 68 patients, and during cholecystectomy in 4 patients. A fast urease test (FUT) to determine the existence of H. pylori in gastric mucosa was carried out for all the patients during the endoscopic examination. The existence of genetic material of H. pylori was determined by detection of ureA gene by the method of nested PCR. The results of this reaction were shown by electrophoresis on 10g.L(-1) agarose gel in a band of 256bp. RESULTS: The majority of the patients included in our study had biliary lithiasis without signs of cholangitis (48 patients, 67%), whereas other patients were complicated by cholangitis (17 patients, 24%). Seven patients (9%) had normal ERCP, forming thus the control group. In the group of patients with lithiasis 26 patients (54.2%) had positive PCR of H. pylori in bile and among the patients with associated cholangitis positive PCR was detected in 9 patients (52.9%). Among the seven patients with normal ERCP only one (14%) had positive PCR of H. pylori. A high percentage of H. pylori infection of gastric mucosa was observed (57 patients, 79%). It was also observed that its slightly higher positivity was in the patients with distinct bile pathology: 81% FUT positive patients in the group with choledocholithiasis alone and 76% in the group with choledocholithiasis associated with cholangitis. Seventy-one percent of the patients with regular findings had positive FUT. CONCLUSION: The prevalence of H. pylori infection both in bile and in gastric mucosa in patients with benign diseases of biliary ducts does not show a statistically significant difference in relation to the prevalence of the same with the patients with normal ERCP. The existence of H. pylori infection possibly does not play a role in pathogenesis of benign biliary diseases.