Advanced Ti-Nb-Ta Alloys for Bone Implants with Improved Functionality.

The additive manufacturing of titanium-niobium-tantalum alloys with nominal chemical compositions Ti-xNb-6Ta (x = 20, 27, 35) by means of laser beam powder bed fusion is reported, and their potential as implant materials is elaborated by mechanical and biological characterization. The properties of dense specimens manufactured in different build orientations and of open porous Ti-20Nb-6Ta specimens are evaluated. Compression tests indicate that strength and elasticity are influenced by the chemical composition and build orientation. The minimum elasticity is always observed in the 90° orientation. It is lowest for Ti-20Nb-6Ta (43.2 ± 2.7 GPa) and can be further reduced to 8.1 ± 1.0 GPa for open porous specimens (p < 0.001). Furthermore, human osteoblasts are cultivated for 7 and 14 days on as-printed specimens and their biological response is compared to that of Ti-6Al-4V. Build orientation and cultivation time significantly affect the gene expression profile of osteogenic differentiation markers. Incomplete cell spreading is observed in specimens manufactured in 0° build orientation, whereas widely stretched cells are observed in 90° build orientation, i.e., parallel to the build direction. Compared to Ti-6Al-4V, Ti-Nb-Ta specimens promote improved osteogenesis and reduce the induction of inflammation. Accordingly, Ti-xNb-6Ta alloys have favorable mechanical and biological properties with great potential for application in orthopedic implants.

Systematic enhancement of microbial decontamination efficiency in bone graft processing by means of high hydrostatic pressure using Escherichia coli as a model organism.

To obtain bone allografts that are safe for transplantation, several processing steps for decellularization and decontamination have to be applied. Currently available processing methods, although well-established, may interfere with the biomechanical properties of the bone. High hydrostatic pressure (HHP) is known to devitalize tissues effectively while leaving the extracellular matrix intact. However, little is known about the inactivation of the contaminating microorganisms by HHP. This study aims to investigate the ability of high-pressure decontamination and to establish a treatment protocol that is able to successfully inactivate microorganisms with the final goal to sterilize bone specimens. Using Escherichia coli (E. coli) as a model organism, HHP treatment parameters like temperature and duration, pressurization medium, and the number of treatment cycles were systematically adjusted to maximize the efficiency of inactivating logarithmic and stationary phase bacteria. Towards that we quantified colony-forming units (cfu) after treatment and investigated morphological changes via Field Emission Scanning Electron Microscopy (FESEM). Additionally, we tested the decontamination efficiency of HHP in bovine cancellous bone blocks that were contaminated with bacteria. Finally, two further model organisms were evaluated, namely Pseudomonas fluorescens as a Gram-negative microorganism and Micrococcus luteus as a Gram-positive representative. A HHP protocol, using 350 MPa, was able to sterilize a suspension of stationary phase E. coli, leading to a logarithmic reduction factor (log RF) of at least -7.99 (±0.43). The decontamination of bone blocks was less successful, indicating a protective effect of the surrounding tissue. Sterilization of 100% of the samples was achieved when a protocol optimized in terms of treatment temperature, duration, pressurization medium, and number and/or interval of cycles, respectively, was applied to bone blocks artificially contaminated with a suspension containing 104 cfu/mL. Hence, we here successfully established protocols for inactivating Gram-negative model microorganisms by HHP of up to 350 MPa, while pressure levels of 600 MPa were needed to inactivate the Gram-positive model organism. Thus, this study provides a basis for further investigations on different pathogenic bacteria that could enable the use of HHP in the decontamination of bone grafts intended for transplantation.

Isolation of TiNbN wear particles from a coated metal-on-metal bearing: Morphological characterization and in vitro evaluation of cytotoxicity in human osteoblasts.

To improve the wear resistance of articulating metallic joint endoprostheses, the surfaces can be coated with titanium niobium nitride (TiNbN). Under poor tribological conditions or malalignment, wear can occur on these implant surfaces in situ. This study investigated the biological response of human osteoblasts to wear particles generated from TiNbN-coated hip implants. Abrasive particles were generated in a hip simulator according to ISO 14242-1/-2 and extracted with Proteinase K. Particle characteristics were evaluated by electron microscopy and energy dispersive x-ray spectroscopy (EDS), inductively coupled plasma mass spectrometry (ICP-MS) and dynamic light scattering (DLS) measurements. Human osteoblasts were exposed to different particle dilutions (1:20, 1:50, and 1:100), and cell viability and gene expression levels of osteogenic markers and inflammatory mediators were analyzed after 4 and 7 days. Using ICP-MS, EDS, and DLS measurements, ~70% of the particles were identified as TiNbN, ranging from 39 to 94 nm. The particles exhibited a flat and subangular morphology. Exposure to particles did not influence cell viability and osteoblastic differentiation capacity. Protein levels of collagen type 1, osteoprotegerin, and receptor activator of nuclear factor κB ligand were almost unaffected. Moreover, the pro-inflammatory response via interleukins 6 and 8 was minor induced after particle contact. A high number of TiNbN wear particles only slightly affected osteoblasts' differentiation ability and inflammatory response compared to metallic particles. Nevertheless, further studies should investigate the role of these particles in peri-implant bone tissue, especially concerning other cell types.

Novel In Vitro Models for Cell Differentiation and Drug Transport Studies of the Human Intestine.

The most common in vitro model for absorption, distribution, metabolism, and excretion (ADME) purposes is currently the Caco-2 cell line. However, clear differences in gene and protein expression towards the small intestine and an, at best, fair prediction accuracy of intestinal drug absorption restrict the usefulness of a model for intestinal epithelial cells. To overcome these limitations, we evaluated a panel of low-passaged patient-derived colorectal cancer cell lines of the HROC collection concerning similarities to small intestinal epithelial cells and their potential to predict intestinal drug absorption. After initial screening of a larger panel, ten cell lines with confluent outgrowth and long-lasting barrier-forming potential were further characterized in close detail. Tight junctional complexes and microvilli structures were detected in all lines, anda higher degree of differentiation was observed in 5/10 cell lines. All lines expressed multiple transporter molecules, with the expression levels in three lines being close to those of small intestinal epithelial cells. Compared with the Caco-2 model, three HROC lines demonstrated both higher similarity to jejunal epithelial tissue cells and higher regulatory potential of relevant drug transporters. In summary, these lines would be better-suited human small intestinal epithelium models for basic and translational research, especially for ADME studies.

A Thia-Analogous Indirubin N-Glycoside Disrupts Mitochondrial Function and Causes the Death of Human Melanoma and Cutaneous Squamous Cell Carcinoma Cells.

Skin cancer is the most common malignant disease worldwide and, therefore, also poses a challenge from a pharmacotherapeutic perspective. Derivatives of indirubin are an interesting option in this context. In the present study, the effects of 3-[3'-oxo-benzo[b]thiophen-2'-(Z)-ylidene]-1-(ß-d-glucopyranosyl)-oxindole (KD87), a thia-analogous indirubin N-glycoside, on the viability and mitochondrial properties of melanoma (A375) and squamous cell carcinoma cells (A431) of the skin were investigated. In both cell lines, KD87 caused decreased viability, the activation of caspases-3 and -7, and the inhibition of colony formation. At the mitochondrial level, a concentration-dependent decrease in both the basal and ATP-linked oxygen consumption rate and in the reserve capacity of oxidative respiration were registered in the presence of KD87. These changes were accompanied by morphological alterations in the mitochondria, a release of mitochondrial cytochrome c into the cytosol and significant reductions in succinate dehydrogenase complex subunit B (SDHB, subunit of complex II) in A375 and A431 cells and NADH:ubiquinone oxidoreductase subunit B8 (NDUFB8, subunit of complex I) in A375 cells. The effect of KD87 was accompanied by a significant upregulation of the enzyme heme oxygenase-1, whose inhibition led to a partial but significant reduction in the metabolic-activity-reducing effect of KD87. In summary, our data show a mitochondria-targeting effect of KD87 as part of the cytotoxic effect of this compound on skin cancer cells, which should be considered in future studies with this class of compounds.

Axenic Long-Term Cultivation of Pneumocystis jirovecii.

Pneumocystis jirovecii, a fungus causing severe Pneumocystis pneumonia (PCP) in humans, has long been described as non-culturable. Only isolated short-term experiments with P. jirovecii and a small number of experiments involving animal-derived Pneumocystis species have been published to date. However, P. jirovecii culture conditions may differ significantly from those of animal-derived Pneumocystis, as there are major genotypic and phenotypic differences between them. Establishing a well-performing P. jirovecii cultivation is crucial to understanding PCP and its pathophysiological processes. The aim of this study, therefore, was to develop an axenic culture for Pneumocystis jirovecii. To identify promising approaches for cultivation, a literature survey encompassing animal-derived Pneumocystis cultures was carried out. The variables identified, such as incubation time, pH value, vitamins, amino acids, and other components, were trialed and adjusted to find the optimum conditions for P. jirovecii culture. This allowed us to develop a medium that produced a 42.6-fold increase in P. jirovecii qPCR copy numbers after a 48-day culture. Growth was confirmed microscopically by the increasing number and size of actively growing Pneumocystis clusters in the final medium, DMEM-O3. P. jirovecii doubling time was 8.9 days (range 6.9 to 13.6 days). In conclusion, we successfully cultivated P. jirovecii under optimized cell-free conditions in a 70-day long-term culture for the first time. However, further optimization of the culture conditions for this slow grower is indispensable.

Effects of prenatal Doppler ultrasound on the retina of the chick embryo in ovo.

Currently, the effect of prenatal ultrasound on foetal development is intensively discussed and the guidelines for prenatal diagnostics have been changed. However, data supporting these concerns are scarce. Therefore, we used an established in ovo model of the chicken embryo to investigate cell proliferation and apoptosis within the retina. A total of 21 chicken eggs were fenestrated on Day 5 and allocated to either the control (n = 8) or exposition group (n = 13). The exposition group was treated with pulsed-wave Doppler ultrasound (PWD) for 10 min while controls remained without treatment. After subsequent incubation (6-48 h), the eggs were sacrificed, and chicken embryos were examined morphologically (HE-staining) and immunohistochemically. Counting of apoptotic and proliferating cells per retina was performed using antibodies specific for phospho-histone-H3 and active caspase-3 in combination with a biotin-labelled secondary antibody and peroxidase conjugated avidin-biotin complex for chromogenic detection. Due to a rather low number of specimens at each time point after ultrasound exposition, we neglected the effects of incubation time and focused on treatment effects. This approach revealed that the median number of proliferating cells is reduced after 10 min of exposure to PWD (569 vs. 766), while the number of apoptotic cells is fairly comparable between groups (5 vs. 6). Our data contribute to a better understanding of prenatal US on foetal development by suggesting that PWD could have an impact on the number of proliferating cells in the developing chicken retina and therefore justify further investigations.

Bsep/Abcb11 knockout ameliorates Schistosoma mansoni liver pathology by reducing parasite fecundity.

BACKGROUND AND AIMS: Schistosoma mansoni infection is one of the worldwide leading causes of liver fibrosis and portal hypertension. The objective of this study was to evaluate whether polyhydroxylated bile acids (BAs), known to protect mice from the development of acquired cholestatic liver injury, counteract S. mansoni-induced inflammation and fibrosis. METHODS: Adult FVB/N wild type (WT) and Abcb11/Bsep-/- mice were infected with either 25 or 50 S. mansoni cercariae. Eight weeks post infection, effects on liver histology, serum biochemistry, gene expression profile of proinflammatory cytokines and fibrotic markers, hepatic hydroxyproline content and FACS analysis were performed. RESULTS: Bsep-/- mice infected with S. mansoni showed significantly less hepatic inflammation and tendentially less fibrosis compared to infected WT mice. Despite elevated alanine aminotransferase, aspartate aminotransferase and alkaline phosphatase levels in infected Bsep-/- mice, inflammatory cells such as M2 macrophages and Mac-2/galectin-3+ cells were reduced in these animals. Accordingly, mRNA-expression levels of anti-inflammatory cytokines (IL-4 and IL-13) were increased in Bsep-/- mice upon infection. Furthermore, infected Bsep-/- mice exhibited decreased hepatic egg load and parasite fecundity, consequently affecting the worm reproduction rate. This outcome could arise from elevated serum BA levels and lower blood pH in Bsep-/- mice. CONCLUSIONS: The loss of Bsep and the resulting changes in bile acid composition and blood pH are associated with the reduction of parasite fecundity, thus attenuating the development of S. mansoni-induced hepatic inflammation and fibrosis.

3D Spheroid Cultivation Alters the Extent and Progression of Osteogenic Differentiation of Mesenchymal Stem/Stromal Cells Compared to 2D Cultivation.

Mesenchymal stem/stromal cells (MSC) are capable of progenitor cell fraction renewal or tissue-specific differentiation. These properties are maintained during in vitro cultivation, making them an interesting model system for testing biological and pharmacological compounds. Cell cultivation in 2D is commonly used to study cellular responses, but the 2D environment does not reflect the structural situation of most cell types. Therefore, 3D culture systems have been developed to provide a more accurate physiological environment in terms of cell-cell interactions. Since knowledge about the effects of 3D culture on specific differentiation processes is limited, we studied the effects on osteogenic differentiation and the release of factors affecting bone metabolism for up to 35 days and compared them with the effects in 2D culture. We demonstrated that the selected 3D model allowed the rapid and reliable formation of spheroids that were stable over several weeks and both accelerated and enhanced osteogenic differentiation compared with the 2D culture. Thus, our experiments provide new insights into the effects of cell arrangement of MSC in 2D and 3D. However, due to the different culture dimensions, various detection methods had to be chosen, which in principle limits the explanatory power of the comparison between 2D and 3D cultures.

Combining Electrostimulation with Impedance Sensing to Promote and Track Osteogenesis within a Titanium Implant.

(1) Background: Electrical stimulation is a promising alternative to promote bone fracture healing but with the limitation of tracking the osteogenesis progress in vivo. To overcome this issue, we present an opportunity to combine the electrical stimulation of a commercial titanium implant, which promotes osteogenesis within the fracture, with a real-time readout of the osteogenic progress by impedance sensing. This makes it possible to adjust the electrical stimulation modalities to the individual patient's fracture healing process. (2) Methods: In detail, osteogenic differentiation of several cell types was monitored under continuous or pulsatile electrical stimulation at 0.7 V AC/20 Hz for at least seven days on a titanium implant by electric cell-substrate impedance sensing (ECIS). For control, chemical induction of osteogenic differentiation was induced. (3) Results: The most significant challenge was to discriminate impedance changes caused by proliferation events from those initiated by osteogenic differentiation. This discrimination was achieved by remodeling the impedance parameter Alpha (α), which increases over time for pulsatile electrically stimulated stem cells. Boosted α-values were accompanied by an increased formation of actin stress fibers and a reduced expression of the focal adhesion kinase in the cell periphery; morphological alterations known to occur during osteogenesis. (4) Conclusions: This work provided the basis for developing an effective fracture therapy device, which can induce osteogenesis on the one hand, and would allow us to monitor the induction process on the other hand.

Decellularization of precision-cut kidney slices-application of physical and chemical methods.

The extracellular matrix (ECM) obtained by decellularization provides scaffolds with the natural complex architecture and biochemical composition of the target organ. Whole kidney decellularization by perfusion uses the vasculature to remove cells leaving a scaffold that can be recellularized with patient-specific cells. However, decellularization and recellularization are highly complex processes that require intensive optimization of various parameters. In pursuit of this, a huge number of animals must be sacrificed. Therefore, we used precision-cut kidney slices (PCKS) as a source of natural scaffolds, which were decellularized by immersion in chemical reagents allowing the examination of more parameters with less animals. However, chemical reagents have a damaging effect on the structure and components of the ECM. Therefore, this study aimed at investigating the effects of physical treatment methods on the effectiveness of PCKS decellularization by immersion in chemical reagents (CHEM). PCKS were treated physically before or during immersion in chemicals (CHEM) with high hydrostatic pressure (HHP), freezing-thawing cycles (FTC) or in an ultrasonic bath system (UBS). Biochemical and DNA quantification as well as structural evaluation with conventional histology and scanning electron microscopy (SEM) were performed. Compared to decellularization by CHEM alone, FTC treatment prior to CHEM was the most effective in reducing DNA while also preserving glycosaminoglycan (GAG) content. Moreover, while UBS resulted in a comparable reduction of DNA, it was the least effective in retaining GAGs. In contrast, despite the pretreatment with HHP with pressures up to 200 MPa, it was the least effective in DNA removal. Histological scoring showed that HHP scaffolds received the best score followed by UBS, FTC and CHEM scaffolds. However further analysis with SEM demonstrated a higher deterioration of the ultrastructure in UBS scaffolds. Altogether, pretreatment with FTC prior to CHEM resulted in a better balance between DNA removal and structural preservation.

MR microscopy of the developing upper extremity of the chicken in ovo using 7 Tesla MRI.

MR microscopy (MRM) is known as ultra-high-field (UHF) magnetic resonance imaging with an in-plane spatial resolution of <100 µm, yields highly resolved non-invasive anatomical imaging and allows longitudinal assessment of embryonic avian development. The aim of the present study was to evaluate the feasibility of in vivo anatomical MRI assessment of the developing upper extremity of the chicken. Thirty-eight fertilized chicken eggs were examined at 7 Tesla acquiring high-resolution T2-weighted images with an in-plane resolution of 74 × 74 µm. To reduce motion artefacts, the eggs were moderately cooled before and during MRI. Development of the upper extremity was anatomically and quantitatively assessed. Chondrification and ossification on MRI were correlated with histological examination. MRM allowed the identification of the embryo from stage D5 onwards. First chondrification of the upper extremity was visible at stage D7, and the differentiation of the forearm was possible from stage D9 throughout the developmental period with excellent correlation to histology. MRM also allowed the differentiation between cortical and medullary bone as well as the detection of chondrified areas. UHF MRM allows the in vivo and in ovo evaluation of the upper limb during embryonic development and provides non-invasive longitudinal anatomical information. This technique allows longitudinal studies of the same embryo during the developmental period and may therefore provide further insights into the development of the upper extremity. With improved coil technique and increasing availability of UHF MR systems, there is great potential regarding several research topics in experimental musculoskeletal radiology.

Experimental Investigation of Material Transfer on Bearings for Total Hip Arthroplasty-A Retrieval Study on Ceramic and Metallic Femoral Heads.

Metallic deposition is a commonly observed phenomenon on the surface of revised femoral heads in total hip arthroplasty and can lead to increased wear due to third bodies. In order to find out the origin and composition of the transfer material, 98 retrieved femoral heads of different materials were examined with regard to the cause of revision, localization, pattern and composition of the transfer material by energy dispersive X-ray spectroscopy. We found that in 53.1%, the deposition was mostly in the region of the equator and the adjacent pole of the femoral heads. The most common cause for revision of heads with metallic deposition was polyethylene wear (43.9%). Random stripes (44.9%), random patches (41.8%) and solid patches (35.7%) were most prevalent on retrieved femoral heads. Random patches were a typical pattern in ceramic-on-ceramic bearing couples. The solid patch frequently occurred in association with dislocation of the femoral head (55%). The elemental analysis of the depositions showed a variety of different materials. In most cases, titanium was an element of the transferred material (76.5%). In addition to metallic components, several non-metallic components were also detected, such as carbon (49%) or sulfur (4.1%). Many of the determined elements could be assigned with regard to their origin with the help of the associated revision cause. Since the depositions lead to an introduction of third-body particles and thus to increased wear, the depositions on the bearing surfaces should be avoided in any case.

The Combination of Δ9-Tetrahydrocannabinol and Cannabidiol Suppresses Mitochondrial Respiration of Human Glioblastoma Cells via Downregulation of Specific Respiratory Chain Proteins.

Phytocannabinoids represent a promising approach in glioblastoma therapy. Previous work has shown that a combined treatment of glioblastoma cells with submaximal effective concentrations of psychoactive Δ9-tetrahydrocannabinol (THC) and non-psychoactive cannabidiol (CBD) greatly increases cell death. In the present work, the glioblastoma cell lines U251MG and U138MG were used to investigate whether the combination of THC and CBD in a 1:1 ratio is associated with a disruption of cellular energy metabolism, and whether this is caused by affecting mitochondrial respiration. Here, the combined administration of THC and CBD (2.5 µM each) led to an inhibition of oxygen consumption rate and energy metabolism. These effects were accompanied by morphological changes to the mitochondria, a release of mitochondrial cytochrome c into the cytosol and a marked reduction in subunits of electron transport chain complexes I (NDUFA9, NDUFB8) and IV (COX2, COX4). Experiments with receptor antagonists and inhibitors showed that the degradation of NDUFA9 occurred independently of the activation of the cannabinoid receptors CB1, CB2 and TRPV1 and of usual degradation processes mediated via autophagy or the proteasomal system. In summary, the results describe a previously unknown mitochondria-targeting mechanism behind the toxic effect of THC and CBD on glioblastoma cells that should be considered in future cancer therapy, especially in combination strategies with other chemotherapeutics.

Implementation of a combined CDK inhibition and arginine-deprivation approach to target arginine-auxotrophic glioblastoma multiforme cells.

Constitutive activation of cyclin-dependent kinases (CDKs) or arginine auxotrophy are hallmarks of Glioblastoma multiforme (GBM). The latter metabolic defect renders tumor cells vulnerable to arginine-depleting substances, such as arginine deiminase from Streptococcus pyogenes (SpyADI). Previously, we confirmed the susceptibility of patient-derived GBM cells towards SpyADI as well as CDK inhibitors (CDKis). To improve therapeutic effects, we here applied a combined approach based on SpyADI and CDKis (dinaciclib, abemaciclib). Three arginine-auxotrophic patient-derived GBM lines with different molecular characteristics were cultured in 2D and 3D and effects of this combined SpyADI/CDKi approach were analyzed in-depth. All CDKi/SpyADI combinations yielded synergistic antitumoral effects, especially when given sequentially (SEQ), i.e., CDKi in first-line and most pronounced in the 3D models. SEQ application demonstrated impaired cell proliferation, invasiveness, and viability. Mitochondrial impairment was demonstrated by increasing mitochondrial membrane potential and decreasing oxygen consumption rate and extracellular acidification rate after SpyADI/abemaciclib monotherapy or its combination regimens. The combined treatment even induced autophagy in target cells (abemaciclib/SpyADI > dinaciclib/SpyADI). By contrast, the unfolded protein response and p53/p21 induced senescence played a minor role. Transmission electron microscopy confirmed damaged mitochondria and endoplasmic reticulum together with increased vacuolization under CDKi mono- and combination therapy. SEQ-abemaciclib/SpyADI treatment suppressed the DSB repair system via NHEJ and HR, whereas SEQ-dinaciclib/SpyADI treatment increased γ-H2AX accumulation and induced Rad51/Ku80. The latter combination also activated the stress sensor GADD45 and ß-catenin antagonist AXIN2 and induced expression changes of genes involved in cellular/cytoskeletal integrity. This study highlights the strong antitumoral potential of a combined arginine deprivation and CDK inhibition approach via complex effects on mitochondrial dysfunction, invasiveness as well as DNA-damage response. This provides a good starting point for further in vitro and in vivo proof-of-concept studies to move forward with this strategy.

Comparative Sample Preparation Using Focused Ion Beam and Ultramicrotomy of Human Dental Enamel and Dentine for Multimicroscopic Imaging at Micro- and Nanoscale.

(1) Background: The aim of this study was to systematically compare TEM sections of mineralized human enamel and dentine prepared by focused ion beam (in situ lift-out) technique and ultramicrotomy through a combination of microscopic examination methods (scanning electron microscopy and transmission electron microscopy). In contrast with published studies, we compared the TEM preparation methods using the same specimen blocks as those for the ultramicrotomy and FIB technique. (2) Methods: A further evaluation of TEM sample preparation was obtained by confocal laser scanning microscopy and atomic force microscopy. In addition, ultramicrotome- and focused ion beam-induced artefacts are illustrated. (3) Results: The FIB technique exposed a major difference between non-decalcified enamel and dentine concerning the ultrastructural morphology compared to ultramicrotome-prepared sections. We found that ultramicrotomy was useful for cutting mineralized dentine, with the possibility of mechanical artefacts, but offers limited options for the preparation of mineralized enamel. FIB preparation produced high-quality TEM sections, showing the anisotropic ultrastructural morphology in detail, with minor structural artefacts. Our results show that the solution of artificial saliva and glutardialdehyde (2.5% by volume) is a very suitable fixative for human mineralized tissue. (4) Conclusions: The protocol that we developed has strong potential for the preparation of mineralized biomaterials for TEM imaging and analysis.

Microbeam Irradiation of the Beating Rodent Heart: An Ex Vivo Study of Acute and Subacute Effects on Cardiac Function.

PURPOSE: Microbeam radiation therapy (MRT) has shown several advantages compared with conventional broad-beam radiation therapy in small animal models, including a better preservation of normal tissue function and improved drug delivery based on a rapidly increased vascular permeability in the target region. Normal tissue tolerance is the limiting factor in clinical radiation therapy. Knowledge of the normal tissue tolerance of organs at risk is therefore a prerequisite in evaluating any new radiation therapy approach. With an irradiation target in the thoracic cavity, the heart would be the most important organ at risk. METHODS AND MATERIALS: We used the ex vivo beating rodent heart in the Langendorff perfusion system at the synchrotron to administer microbeam irradiation (MBI) with a peak dose of 40 or 400 Gy. By continuously recording the electrocardiogram, the left ventricular pressure, and the aortic pressure before, during and after MBI, we were able to assess acute and subacute effects of MBI on electrophysiological and mechanical cardiac function. In addition, we analyzed histologic and ultrastructural sequelae caused by MBI. RESULTS: There were no significant changes in heart rate, heart rate variability, systolic increase of left ventricular pressure or aortic pressure. Moreover, the changes of heart rate, left ventricular pressure and aortic pressure by adding 10-5 mol/L norepinephrine to the perfusate, were also not significant between MBI and sham experiments. However, the rate-pressure product as a surrogate marker for maximum workload after MBI was significantly lower compared with sham-irradiated controls. On the structural level, no severe membranous, sarcomeric, mitochondrial or nuclear changes caused by MBI were detected by desmin immunohistochemistry and electron microscopy. CONCLUSIONS: With respect to acute and subacute toxicity, an MBI peak dose up to 400 Gy did not result in severe changes in cardiac electrophysiology or mechanics.

A Comparative Study on the Adipogenic Differentiation of Mesenchymal Stem/Stromal Cells in 2D and 3D Culture.

Mesenchymal stem/stromal cells (MSC) are capable of renewing the progenitor cell fraction or differentiating in a tissue-specific manner. Adipogenic differentiation of adipose-tissue-derived MSC (adMSC) is important in various pathological processes. Adipocytes and their progenitors are metabolically active and secrete molecules (adipokines) that have both pro- and anti-inflammatory properties. Cell culturing in 2D is commonly used to study cellular responses, but the 2D environment does not reflect the structural situation for most cell types. Therefore, 3D culture systems have been developed to create an environment considered more physiological. Since knowledge about the effects of 3D cultivation on adipogenic differentiation is limited, we investigated its effects on adipogenic differentiation and adipokine release of adMSC (up to 28 days) and compared these with the effects in 2D. We demonstrated that cultivation conditions are crucial for cell behavior: in both 2D and 3D culture, adipogenic differentiation occurred only after specific stimulation. While the size and structure of adipogenically stimulated 3D spheroids remained stable during the experiment, the unstimulated spheroids showed signs of disintegration. Adipokine release was dependent on culture dimensionality; we found upregulated adiponectin and downregulated pro-inflammatory factors. Our findings are relevant for cell therapeutic applications of adMSC in complex, three-dimensionally arranged tissues.

Cuprizone Intoxication Results in Myelin Vacuole Formation.

Myelin damage is a histopathological hallmark of multiple sclerosis lesions. Results of post mortem studies suggest that impaired myelin-axon interaction characterized by focal myelin detachments is an early event during lesion genesis. In this study, we investigated the ultrastructural changes of the axon-myelin interface in the cuprizone model using serial block face scanning electron microscopy and immunohistochemistry. We show that non-inflammatory injury of oligodendrocytes by cuprizone intoxication results in myelin vacuole formation and axonal swellings, paralleled by early alterations of the node of Ranvier cytoarchitecture. This remarkable resemblance of ultrastructural myelin characteristics in multiple sclerosis and the cuprizone animal model suggests that the cuprizone model is a valuable tool to study early pathologies during lesion formation.

BMP-2 Long-Term Stimulation of Human Pre-Osteoblasts Induces Osteogenic Differentiation and Promotes Transdifferentiation and Bone Remodeling Processes.

Bone morphogenic protein (BMP-) 2 plays an important role in the regeneration of bone defects by promoting osteogenic differentiation. However, several animal studies have reported adverse side effects of BMP-2, including osteoclast activation, induction of peroxisome proliferator- activated receptor gamma (PPARG)expression, and inflammation. High BMP-2 concentrations are thought to be responsible for these side effects. For this reason, primary pre-osteoblasts were exposed to lower BMP-2 concentrations (1 and 2 µg/mL). Long-term exposure (up to 28 days) was performed to investigate whether this stimulation protocol may promote osteogenic differentiation without causing the side effects mentioned above. The results showed that BMP-2 treatment for 14 or 28 days resulted in increased osteogenesis, through an increase in runt-related transcription factor 2, osterix, alkaline phosphatase, and integrin-binding sialoprotein expression. However, an increase in tumor necrosis factor alpha and receptor activator of nuclear factor kappa-Β ligand protein levels was observed after BMP-2 exposure, indicating also an increased potential for osteoclast activation by osteoblasts. Additionally, morphological changes like intracellular, filled vacuoles could be detected. Enhanced PPARG and perilipin 1 mRNA transcripts and lipid droplets indicated an induced adipogenic differentiation. Overall, the data demonstrate that long-term BMP-2 exposure promotes not only osteogenic differentiation but also adipogenesis and regulates mediators involved in osteoclast activation in vitro.