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Arteriosclerosis with its clinical sequelae (cardiac infarction, stroke, peripheral arterial occlusive disease) and vascular/Alzheimer dementia not only result in far more than half of all deaths but also represent dramatic economic problems. The reason is, among others, that diabetes mellitus is an independent risk factor for both disorders, and the number of diabetics strongly increases worldwide. More than one-half of infants in the first 6 months of life have already small collections of macrophages and macrophages filled with lipid droplets in susceptible segments of the coronary arteries. On the other hand, the authors of the Bogalusa Heart Study found a strong increase in the prevalence of obesity in childhood that is paralleled by an increase in blood pressure, blood lipid concentration, and type 2 Diabetes mellitus. Thus, there is a clear linkage between arteriosclerosis/Alzheimer's disease on the one hand and diabetes mellitus on the other hand. Furthermore, it has been demonstrated that distinct apoE isoforms on the blood lipids further both arteriosclerotic and Alzheimer nanoplaque formation and therefore impair flow-mediated vascular reactivity as well. Nanoplaque build-up seems to be the starting point for arteriosclerosis and Alzheimer's disease in their later full clinical manifestation.
In earlier work, we could portray the anionic biopolyelectrolytes syndecan/perlecan as blood flow sensors and lipoprotein receptors in cell membrane and vascular matrix. We described extensively molecular composition, conformation, form and function of the macromolecule heparan sulfate proteoglycan (HS-PG). In two supplementary experimental settings (ellipsometry, myography),we utilized isolated HS-PG for in vitro nanoplaque investigations and isolated human coronary artery segments for in vivo tension measurements.
With the ellipsometry-based approach, we were successful in establishing a direct connection on a molecular level between diabetes mellitus on the one side and arteriosclerosis/Alzheimer's disease on the other side. Application of glucose at a concentration representative for diabetics and leading to glycation of proteins and lipids, entailed a significant increase in arteriosclerotic and Alzheimer nanoplaque formation. IDLapoE4/E4 was by far superior to IDLapoE3/E3 in Plaque build-up, both in diabetic and non-diabetic patients. Recording vascular tension of flow-dependent reactivity in blood substitute solution and under application of different IDLapoE isoforms showed an impaired vasorelaxation for pooled IDL and IDLapoE4/E4, thus confirming the ellipsometric investigations. Incubation in IDLapoE0/E0 (apoE “knockout man”), however, resulted in a massive flowmediated contraction, also complemented by strongly aggregated nanoplaques.
In contrast, HDL was shown to present a powerful protection against nanoplaque formation on principle, both in the in vitro model and the in vivo scenario on the endothelial cell membrane. The competitive interplay with LDL is highlighted through the flow experiment, where flow-mediated, HDL-induced vasodilatation remains untouched by additional incubation with LDL. This is due to the four times higher affinity for the proteoglycan receptor of HDL as compared to LDL.
Taken together, the studies demonstrate that while simplistic, the ellipsometry approach and the endothelialmimicking proteoglycan-modified surfaces provide information on the initial steps of lipoprotein-related Plaque formation, which correlates with findings on endothelial cells and blood vessels, and afford insight into the role of lipoprotein deposition and Exchange phenomena at the onset of these pathophysiologies.
Living organisms constantly interact with their environment, including through the exchange of gases. Respiration and the release and uptake of volatile organic compounds (VOCs) create dynamic microenvironments in biological systems. Studying the kinetics of volatiles in biological systems requires expensive instruments, and data analysis is challenging. Therefore, we aimed to design a minimal analytical device for measuring the composition of gaseous mixtures in realtime.
We built the ‘Modular Biological Mass Spectrometer’ (MoBiMS) from 3D-printed parts and custom sensors to fit a wide array of experimental set-ups. We tested the chemical detection range and temporal resolution of the MoBiMS employing pure compounds and complex biological samples.
Compounds with a higher than 0.4 mmHg vapor pressure and a molecular weight up to 154 g/mol were reliably sensed within seconds. The generated electron impact (EI) spectra were directly comparable with standard databases like the NIST EI library. Under a direct analysis approach, the MoBiMS identified the characteristic odor of banana (Musa sp.), that is, isoamyl acetate; tracked the dynamics of CO2 release while the Alka-Seltzer® reaction occurred showed the kinetics of the transient production and consumption of carbon dioxide during photosynthesis. MoBiMS also discriminated between volatile compounds ions coming from tobacco (Nicotiana benthamiana) leaves and the surrounding air through untargeted analysis. The small footprint of the MoBiMS and its relatively low energy consumption facilitate in situ analyses, as an additional gas supply is not necessary with EI ionization. The MoBiMS is easy to assemble, and its construction and operation are very cost-efficient compared to commercial devices. The analytical performance of the MoBiMS is suitable for real-time studies of biological systems, environmental monitoring, and medical diagnostics.
Osteoclastic bioresorption of biomaterials: Two- and three-dimensional imaging and quantification
(2010)
Purpose: Bioresorbable materials have been developed in the hope that the body will replace them with newly formed tissue. The first step of this remodeling process in bone is the bioresorption of the material by osteoclasts. The aim of this study was to analyze osteoclastic resorption of biomaterials in vitro using the commonly used two-dimensional methods of light-microscopy (LM) and scanning electron microscopy (SEM) in comparison with infinite focus microscopy (IFM), a recently developed imaging method allowing for three-dimensional surface analysis. Methods: Human hematopoietic stem cells were cultivated in the presence of the cytokines M-CSF and RANK-L for 4 weeks directly on dentin and a calcium phosphate cement. Osteoclast development was surveyed with standard techniques. After removal of the cells, resorption was characterized and quantified by LM, SEM and IFM. Results: Osteoclast cultures on the biomaterials presented the typical osteoclast-specific markers. On dentin samples LM, SEM as well as IFM allowed for discrimination of resorption. Quantification of the resorbed area showed a linear correlation between the results (LM vs. SEM: r=0.996, p=0.004; SEM vs. IFM: r=0.989, p=0.011; IFM vs. LM: r=0.995). It was not possible to demarcate resorption pits on GB14 using LM or SEM. With IFM, resorption on GB14 could be visualized and quantified two- and three-dimensionally.
Commonly, to determine osteoclastic resorption of biomaterials only the resorbed area is measured. The depth of the resorption pit, however, may also be important for the performance of a material. To generate such data we used two calcium phosphate ceramics (Ca10 and Ca2). The solubility of the materials was determined according to DIN EN ISO 10993-14. They were scanned three-dimensionally using infinite focus microscopy and subsequently cultivated for 4 weeks in simulated body fluid without (control) or with human osteoclasts. After this cultivation period osteoclasts number was determined and surface changes were evaluated two- and three-dimensionally. Ca10 and Ca2 showed solubilities of 11.0 ± 0.5 and 23.0 ± 2.2 mg g-1, respectively. Both materials induced a significant increase in osteoclast number. While Ca10 did not show osteoclastic resorption, Ca2 showed an increased pit area and pit volume due to osteoclastic action. This was caused by an increased average pit depth and an increased number of pits, while the average area of single pits did not change significantly. The deduced volumetric osteoclastic resorption rate (vORR) of Ca2 (0.010.02 µm3 µm-2 day-1) was lower than the remodelling speed observed in vivo (0.08 µm3 µm-2 day-1), which is in line with the observation that implanted resorbable materials remain in the body longer than originally expected. Determination of volumetric indices of osteoclastic resorption might be valuable in obtaining additional information about cellular resorption of bone substitute materials. This may help facilitate the development of novel materials for bone substitution.
Nanotechnology has enabled the discovery of a multitude of novel materials exhibiting unique physicochemical (PChem) properties compared to their bulk analogues. These properties have led to a rapidly increasing range of commercial applications; this, however, may come at a cost, if an association to long-term health and environmental risks is discovered or even just perceived. Many nanomaterials (NMs) have not yet had their potential adverse biological effects fully assessed, due to costs and time constraints associated with the experimental assessment, frequently involving animals. Here, the available NM libraries are analyzed for their suitability for integration with novel nanoinformatics approaches and for the development of NM specific Integrated Approaches to Testing and Assessment (IATA) for human and environmental risk assessment, all within the NanoSolveIT cloud-platform. These established and well-characterized NM libraries (e.g. NanoMILE, NanoSolutions, NANoREG, NanoFASE, caLIBRAte, NanoTEST and the Nanomaterial Registry (>2000 NMs)) contain physicochemical characterization data as well as data for several relevant biological endpoints, assessed in part using harmonized Organisation for Economic Co-operation and Development (OECD) methods and test guidelines.
Integration of such extensive NM information sources with the latest nanoinformatics methods will allow NanoSolveIT to model the relationships between NM structure (morphology), properties and their adverse effects and to predict the effects of other NMs for which less data is available. The project specifically addresses the needs of regulatory agencies and industry to effectively and rapidly evaluate the exposure, NM hazard and risk from nanomaterials and nano-enabled products, enabling implementation of computational ‘safe-by-design’ approaches to facilitate NM commercialization.