Anja Henss

Applicability of ToF-SIMS for monitoring compositional changes in bone in a long-term animal model

Time-of-flight secondary ion mass spectrometry (ToF-SIMS) is a well-established technique in material sciences but has not yet been widely explored for implementation in life sciences. Here, we demonstrate the applicability and advantages of ToF-SIMS analysis for the study of minerals and biomolecules in osseous tissue. The locally resolved analysis of fragment ions deriving from the sample surface enables imaging and differentiation of bone tissue and facilitates histology on non-stained cross sections. In a rat model, bilateral ovariectomy combined with either a multi-deficiency diet or steroid treatment was carried out to create osteoporotic conditions. We focused our study on the Ca content of the mineralized tissue and monitored its decline. Calcium mass images of cross sections show the progressive degenerative changes in the bone. We observed a decreased Ca concentration in the edge region of the trabeculae and a decline in the Ca/P ratio. Additionally, we focused on the non-mineralized matrix and identified fragment ions that are characteristic for the collagen matrix. We observed trabeculae with wide ranges of non-mineralized collagen for the diet group owing to an impaired mineralization process. Here, the advantage of coeval monitoring of collagen and minerals indicated an osteomalacic model rather than an osteoporotic one.

Impaired extracellular matrix structure resulting from malnutrition in ovariectomized mature rats

Abstract Bone loss is a symptom related to disease and age, which reflects on bone cells and ECM. Discrepant regulation affects cell proliferation and ECM localization. Rat model of osteoporosis (OVX) was investigated against control rats (Sham) at young and old ages. Biophysical, histological and molecular techniques were implemented to examine the underlying cellular and extracellular matrix changes and to assess the mechanisms contributing to bone loss in the context of aging and the widely used osteoporotic models in rats. Bone loss exhibited a compromised function of bone cells and infiltration of adipocytes into bone marrow. However, the expression of genes regulating collagen catabolic process and adipogenesis was chronologically shifted in diseased bone in comparison with aged bone. The data showed the involvement of Wnt signaling inhibition in adipogenesis and bone loss due to over-expression of SOST in both diseased and aged bone. Further, in the OVX animals, an integrin-mediated ERK activation indicated the role of MAPK in osteoblastogenesis and adipogenesis. The increased PTH levels due to calcium and estrogen deficiency activated osteoblastogenesis. Thusly, RANKL-mediated osteoclastogenesis was initiated. Interestingly, the data show the role of MEPE regulating osteoclast-mediated resorption at late stages in osteoporotic bone. The interplay between ECM and bone cells change tissue microstructure and properties. The involvement of Wnt and MAPK pathways in activating cell proliferation has intriguing similarities to oncogenesis and myeloma. The study indicates the importance of targeting both pathways simultaneously to remedy metabolic bone diseases and age-related bone loss.

Time of flight secondary ion mass spectrometry of bone—Impact of sample preparation and measurement conditions

Time of flight secondary ion mass spectrometry (ToF-SIMS) enables the simultaneous detection of organic and inorganic ions and fragments with high mass and spatial resolution. Due to recent technical developments, ToF-SIMS has been increasingly applied in the life sciences where sample preparation plays an eminent role for the quality of the analytical results. This paper focusses on sample preparation of bone tissue and its impact on ToF-SIMS analysis. The analysis of bone is important for the understanding of bone diseases and the development of replacement materials and new drugs for the cure of diseased bone. The main purpose of this paper is to find out which preparation process is best suited for ToF-SIMS analysis of bone tissue in order to obtain reliable and reproducible analytical results. The influence of the embedding process on the different components of bone is evaluated using principal component analysis. It is shown that epoxy resin as well as methacrylate based plastics (Epon and Technovit) as embedding materials do not infiltrate the mineralized tissue and that cut sections are better suited for the ToF-SIMS analysis than ground sections. In case of ground samples, a resin layer is smeared over the sample surface due to the polishing step and overlap of peaks is found. Beside some signals of fatty acids in the negative ion mode, the analysis of native, not embedded samples does not provide any advantage. The influence of bismuth bombardment and O2 flooding on the signal intensity of organic and inorganic fragments due to the variation of the ionization probability is additionally discussed. As C60 sputtering has to be applied to remove the smeared resin layer, its effect especially on the organic fragments of the bone is analyzed and described herein.

Storage of cell samples for ToF-SIMS experiments—How to maintain sample integrity

In order to obtain comparable and reproducible results from time-of-flight secondary ion mass spectrometry (ToF-SIMS) analysis of biological cells, the influence of sample preparation and storage has to be carefully considered. It has been previously shown that the impact of the chosen preparation routine is crucial. In continuation of this work, the impact of storage needs to be addressed, as besides the fact that degradation will unavoidably take place, the effects of different storage procedures in combination with specific sample preparations remain largely unknown. Therefore, this work examines different wet (buffer, water, and alcohol) and dry (air-dried, freeze-dried, and critical-point-dried) storage procedures on human mesenchymal stem cell cultures. All cell samples were analyzed by ToF-SIMS immediately after preparation and after a storage period of 4 weeks. The obtained spectra were compared by principal component analysis with lipid- and amino acid-related signals known from the literature. In all dry storage procedures, notable degradation effects were observed, especially for lipid-, but also for amino acid-signal intensities. This leads to the conclusion that dried samples are to some extent easier to handle, yet the procedure is not the optimal storage solution. Degradation proceeds faster, which is possibly caused by oxidation reactions and cleaving enzymes that might still be active. Just as well, wet stored samples in alcohol struggle with decreased signal intensities from lipids and amino acids after storage. Compared to that, the wet stored samples in a buffered or pure aqueous environment revealed no degradation effects after 4 weeks. However, this storage bears a higher risk of fungi/bacterial contamination, as sterile conditions are typically not maintained. Thus, regular solution change is recommended for optimized storage conditions. Not directly exposing the samples to air, wet storage seems to minimize oxidation effects, and hence, buffer or water storage with regular renewal of the solution is recommended for short storage periods.

Small changes in bone structure of female α7 nicotinic acetylcholine receptor knockout mice

BackgroundRecently, analysis of bone from knockout mice identified muscarinic acetylcholine receptor subtype M3 (mAChR M3) and nicotinic acetylcholine receptor (nAChR) subunit α2 as positive regulator of bone mass accrual whereas of male mice deficient for α7-nAChR (α7KO) did not reveal impact in regulation of bone remodeling. Since female sex hormones are involved in fair coordination of osteoblast bone formation and osteoclast bone degradation we assigned the current study to analyze bone strength, composition and microarchitecture of female α7KO compared to their corresponding wild-type mice (α7WT).MethodsVertebrae and long bones of female 16-week-old α7KO (n = 10) and α7WT (n = 8) were extracted and analyzed by means of histological, radiological, biomechanical, cell- and molecular methods as well as time of flight secondary ion mass spectrometry (ToF-SIMS) and transmission electron microscopy (TEM).ResultsBone of female α7KO revealed a significant increase in bending stiffness (p < 0.05) and cortical thickness (p < 0.05) compared to α7WT, whereas gene expression of osteoclast marker cathepsin K was declined. ToF-SIMS analysis detected a decrease in trabecular calcium content and an increase in C4H6N+ (p < 0.05) and C4H8N+ (p < 0.001) collagen fragments whereas a loss of osteoid was found by means of TEM.ConclusionsOur results on female α7KO bone identified differences in bone strength and composition. In addition, we could demonstrate that α7-nAChRs are involved in regulation of bone remodelling. In contrast to mAChR M3 and nAChR subunit α2 the α7-nAChR favours reduction of bone strength thereby showing similar effects as α7β2-nAChR in male mice. nAChR are able to form heteropentameric receptors containing α- and β-subunits as well as the subunits α7 can be arranged as homopentameric cation channel. The different effects of homopentameric and heteropentameric α7-nAChR on bone need to be analysed in future studies as well as gender effects of cholinergic receptors on bone homeostasis.

ToF-SIMS study of differentiation of human bone-derived stromal cells: new insights into osteoporosis

AbstractLipids have numerous important functions in the human body, as they form the cells’ plasma membranes and play a key role in many disease states, presumably also in osteoporosis. Here, the fatty acid composition of the outer plasma membranes of cells differentiated into the osteogenic and adipogenic direction is studied with surface-sensitive time-of-flight secondary ion mass spectrometry (ToF-SIMS). For data evaluation, principal component analysis (PCA) is applied. Human (bone-derived) mesenchymal stromal cells (hMSCs) from an osteoporotic donor and a control donor are compared to reveal differences in the fatty acid composition of the membranes. The chemical information is correlated to staining and real-time quantitative polymerase chain reaction (rt-qPCR) results to provide insight into the gene expression of several differentiation markers on the RNA level. Adipogenic differentiation of hMSCs from a non-osteoporotic donor correlates with increased relative intensities of all fatty acids under investigation. After osteogenic differentiation of non-osteoporotic cells, the relative mass signal intensities of unsaturated fatty acids such as oleic and linoleic acids are increased. However, the osteoporotic cells show increased levels of palmitic acid in the plasma membrane after exposure to osteogenic differentiation conditions, which correlates to an immature differentiation state relative to non-osteoporotic osteogenic cells. This immature differentiation state is confirmed by increased early osteogenic differentiation factor Runx2 on RNA level and by less calcium mineralization spots seen in von Kossa staining and ToF-SIMS images. Graphical abstractTime-of-flight secondary ion mass spectrometry is applied to analyze the fatty acid composition of the outer plasma membranes of cells differentiated into the adipogenic and osteogenic direction. Cells from an osteoporotic and a control donor are compared to reveal differences due to differentiation and disease stage of the cells.

High resolution imaging and 3D analysis of Ag nanoparticles in cells with ToF-SIMS and delayed extraction

Within this study, the authors use human mesenchymal stem cells incubated with silver nanoparticles (AgNPs) as a model system to systematically investigate the advantages and drawbacks of the fast imaging delayed extraction mode for two-dimensional and three-dimensional (3D) analyses at the cellular level. The authors compare the delayed extraction mode with commonly employed measurement modes in terms of mass and lateral resolution, intensity, and dose density. Using the delayed extraction mode for single cell analysis, a high mass resolution up to 4000 at m/z = 184.08 combined with a lateral resolution up to 360 nm is achieved. Furthermore, the authors perform 3D analyses with Ar-clusters (10 keV) and O2+ (500 eV) as sputter species, combined with Bi3+ and delayed extraction for analysis. Cell compartments like the nucleus are visualized in 3D, whereas no realistic 3D reconstruction of intracellular AgNP is possible due to the different sputter rates of inorganic and organic cell materials. Furthermore, the authors show that the sputter yield of Ag increases with the decreasing Ar-cluster size, which might be an approach to converge the different sputter rates.

Imaging of Lipids in Native Human Bone Sections Using TOF–Secondary Ion Mass Spectrometry, Atmospheric Pressure Scanning Microprobe Matrix-Assisted Laser Desorption/Ionization Orbitrap Mass Spectrometry, and Orbitrap–Secondary Ion Mass Spectrometry

A method is described for high-resolution label-free molecular imaging of human bone tissue. To preserve the lipid content and the heterogeneous structure of osseous tissue, 4 μm thick human bone sections were prepared via cryoembedding and tape-assisted cryosectioning, circumventing the application of organic solvents and a decalcification step. A protocol for comparative mass spectrometry imaging (MSI) on the same section was established for initial analysis with time-of-flight secondary ion mass spectrometry (TOF-SIMS) at a lateral resolution of 10 μm to <500 nm, followed by atmospheric pressure scanning microprobe matrix-assisted laser desorption/ionization (AP-SMALDI) Orbitrap MSI at a lateral resolution of 10 μm. This procedure ultimately enabled MSI of lipids, providing the lateral localization of major lipid classes such as glycero-, glycerophospho-, and sphingolipids. Additionally, the applicability of the recently emerged Orbitrap-TOF-SIMS hybrid system was exemplarily examined and compared to the before-mentioned MSI methods.

Biomaterials-Potential nucleation agents in blood and possible implications.

Blood, simulated body fluids, and many cell culture media are supersaturated solutions with respect to several calcium phosphates. Therefore biomaterials can act as nucleation agents and evoke heterogeneous nucleation of salts on the surface of immersed biomaterials. Depending on the field of application, this can be either beneficial or disadvantageous. Although nucleation from supersaturated solutions is an old and well-known scientific phenomenon it is not standard to test new developed materials with surface analytical methods for their ability to initiate nucleation in vitro. Therefore, this communication aims to review the mineralization effect and to emphasize the possible negative implications, especially to functionalized bone implants. Surface coatings with proteins, growth factors, and, etc., can become ineffective due to deposition of a dense calcium phosphate layer. In the case of drug loaded implants, drug release might be inhibited.

Transition metal complexes with cage-opened diamondoid tetracyclo[7.3.1.14,12.02,7]tetradeca-6.11-diene

Cage-opened diamondoid tetracyclo[7.3.1.14,12.02,7]tetradeca-6,11-diene forms complexes with AgNO3 and CuCl. The latter crystallized from acetonitrile in polymeric form [Cu2Cl2(CH3CN)(diene)]n; in the presence of 2,2′-bipyridine, a double-charged monomeric Cu(I)-complex [Cu2(bipy)2(diene)]2+ formed. Both complexes were structurally characterized through X-ray crystal diffraction analysis. Graphical abstract Copper(I) complexes with the diamantane diene tetracyclo[7.3.1.14,12.02,7]tetradeca-6.11-diene (6) have been prepared and an interesting crystal structure, [Cu2(bipy)2(6)]2+ (hydrogen atoms, anions, and solvent molecules are omitted for clarity), was obtained.