Gerhard Fulda

Structural alterations of adhesion mediating components in cells cultured on poly-β-hydroxy butyric acid

Polymers may serve as a biodegradable material in tissue engineering. To assess the biocompatibility of poly-beta-hydroxy butyric acid (PHB), we studied the structural organization of cellular molecules involved in adhesion using osteoblastic and epithelial cell lines. On PHB, both cell lines revealed a rounded cell shape due to reduced spreading. The filamentous organization of the actin cytoskeleton was impaired. In double immunofluorescence analyses we demostrated that the colocalization of the fibronectin fibrils with the actin filaments was lost in cultures on PHB. Similarly, collagen II distribution was altered, whereas the organization of collagen I was not obviously affected. Further evidence for impaired structural organization was obtained for the beta1-integrin receptor and vinculin which mediate the interaction of the cytoskeleton with the extracellular matrix. In confluent epithelial cells, the tight junction protein ZO-1 showed a larger lateral extension in the cell-cell contacts when cells were grown on PHB. Because structural organization of components which mediate cell-matrix and cell-cell adhesion controls cell physiology these parameters could be a sensitive indicator for the biocompatibility of implant materials.

Biodegradation of titanium implants after long-time insertion used for the treatment of fractured upper and lower jaws through osteosynthesis: element analysis by electron microscopy and EDX or EELS.

Twelve patients underwent an osteosynthesis with titanium to treat upper and lower jaw fractures. Six to 12 months later, the miniplates were removed. Tissue samples were analyzed by light and electron microscopy for detection of a metallosis. The analysis showed new bone formation like callus tissue around the miniplates. In some cases small, rounded deposits and accumulation of colloid-like particles located next to bigger titanium artifacts were detected in the cytoplasm of histiocytes and in the matrix of connective tissue. The titanium was identified by elemental analysis using EDX in SEM as well as by EELS and electron diffraction in TEM. Both kinds of particles contain titanium, but they seem to be different in composition and derivation. The bigger particles seem to consist of metallic titanium and sourced by working on the metallic implants during the implantation itself. On the other hand, the colloidal-like, small, rounded particles in tissue macrophages and outside the cells in the matrix of connective tissue are presumably of other origin; for example, they could be derived from biodegradation and chemical conversion of the metallic implants. The titanium miniplates were examined before and after implantation by using ESCA technique and revealed metallic titanium and different compositions with other elements. The amount of titanium load of the tissue was very low in most cases and presumably not of biomedical relevance.Twelve patients underwent an osteosynthesis with titanium to treat upper and lower jaw fractures. Six to 12 months later, the miniplates were removed. Tissue samples were analyzed by light and electron microscopy for detection of a metallosis. The analysis showed new bone formation like callus tissue around the miniplates. In some cases small, rounded deposits and accumulation of colloid-like particles located next to bigger titanium artifacts were detected in the cytoplasm of histiocytes and in the matrix of connective tissue. The titanium was identified by elemental analysis using EDX in SEM as well as by EELS and electron diffraction in TEM. Both kinds of particles contain titanium, but they seem to be different in composition and derivation. The bigger particles seem to consist of metallic titanium and sourced by working on the metallic implants during the implantation itself. On the other hand, the colloidal-like, small, rounded particles in tissue macrophages and outside the cells in the matrix of connective tissue are presumably of other origin; for example, they could be derived from biodegradation and chemical conversion of the metallic implants. The titanium miniplates were examined before and after implantation by using ESCA technique and revealed metallic titanium and different compositions with other elements. The amount of titanium load of the tissue was very low in most cases and presumably not of biomedical relevance.

Electron Microscopic Detection of Copper in the Liver of Two Patients with Morbus Wilson by EELS and EDX

A 20-year-old male patient with morbus Wilson was liver transplanted because of terminal failure of liver function. The explanted liver showed a strong macronodular cirrhosis as typically seen in Wilson disease. There were visible granular accumulations in the hepatocytes after the rubeanic acid or rhodanine method for histochemical detection of copper. The electron microscopic studies on ultrathin sections revealed numerous electron-dense lysosomes and residual bodies. The elemental analysis in transmission electron microscope (TEM) with electron energy loss spectroscopy (EELS) and in scanning electron microscope (SEM) with energy dispersive x-ray analysis (EDX) showed copper-specific signals of electron-dense accumulations inside these dark lysosomes and residual bodies. In a second case, Wilson disease was diagnosed after autopsy of a 31-year-old patient by liver electron microscopy and EELS; strong electron-dense lysosomes and residual bodies with positive copper signals were found inside hepatocytes. For negative control, hepatocytes with iron accumulation after idiopathic hemochromatosis and liver cirrhosis were also analyzed by EELS in TEM, which showed strong iron, but only a few or no copper signals. Atomic absorption spectroscopy (AAS) in 16 liver samples of healthy and cirrhotic liver revealed only in both cases of Wilson disease a strong increased copper concentration higher than 100 µg Cu/g. The electron microscopic detection of copper-containing hepatocytic lysosomes is helpful for the diagnosis of early stages of Wilson disease in addition to the quantification of hepatic copper by AAS.

Flow cytometric measurements of intracellular Ca2+ mobilization in isolated rat pancreatic acinar cells after hormone stimulation and action of lectins

Isolated rat pancreatic acinar cells were loaded with the Ca(2+)-sensitive fluorescence dye Fluo 3 in vitro and the intracellular Ca2+ changes were analysed by flow cytometry. Morphology, viability, and loading with the dye were studied by light microscopy. Stimulation with cholecystokinin/pancreozymin (CCK) and its agonist caerulein as well as with carbamylcholine (Jestryl) led to an increase of intracellular calcium ions and a fluorescence peak. The slope and height of the Ca2+ signals were found to be influenced by preincubation of cells with some plant lectins (WGA, UEA, PHA, Con A, LCA, PNA). These effects are discussed with respect to the interaction of lectins with the carbohydrate chains of cell membrane receptors.

Electron Microscopic Detection of Tin Accumulation in Biliopancreatic Concrements after Induction of Chronic Pancreatitis in Rats by Di- n -butyltin Dichloride

The organotin compound di- n -butyltin dichloride (DBTC) is able to induce an acute and later a chronic pancreatitis in rats. In previous papers the authors demonstrated this DBTC pancreatitis as a rat model for an interstitial pancreatitis with tendency to transduction to the chronic form. DBTC is excreted according to its lipophilic nature by liver and bile. Therefore, the bilio-pancreatic main duct is necrotized by the tin-loaded bile. The duct system is blocked by cell debris and later by epithelial proliferations. In the chronic phase, numerous rats develop concrements in the main duct. In the present paper, the authors report about bacterial growth in some bilio-pancreatic concrements. Whereas the electron microscopic detection of tin by energy-dispersive X-ray analysis (EDX) in SEM or electron energy loss spectroscopy (EELS) in TEM was negative in the parenchyma of pancreas and liver, some concrements with bacterial cells were positive for this element. Tin mapping with energy spectroscopic imaging (ESI) in TEM demonstrated the congruency of tin signals and electron-dense particles inside these bacteria and of electron-dense accumulations in the matrix of these concrements. The low content of tin in pancreatic and liver tissue and the higher quantity of tin inside the bacterial contaminated concrements were supported by atomic absorption spectrophotometry (AAS). The paper discusses the long time preservation of tin in the concrements as an action of heavy-metal-accumulating bacteria, which should be classified in the future by bacteriological methods.

Hereditary hemochromatosis of a young girl: detection of early iron deposition in liver cell lysosomes using transmission electron microscopy and electron energy loss spectroscopy.

A 14-year-old girl demonstrated increased iron concentration and transferrin saturation, suggesting iron overload of unknown origin. Liver biopsy showed no fibrosis or hepatocytic atrophia. Nevertheless, Prussian blue reaction for histochemical detection of iron demonstrated very weak positive granules in a few hepatocytes on the periphery of hepatic lobules in close connection to bile capillaries. This very early stage of hemochromatosis was confirmed by TEM and EELS for iron accumulation inside hepatocytic lysosomes and residual bodies. Such siderosomes were scarce in number and iron content, compared to a case of manifested hemochromatosis and liver cirrhosis (Jonas L, Fulda G, Salemeh T, et al. Ultrastruct Pathol. 2001; 25: 111-118.). Liver iron concentration as measured by inductively coupled plasma-mass spectrometry (ICP-MS) and atomic absorption spectrometry (AAS) yielded 2.005 mg/g tissue dry weight, which was considered not significantly increased. In the absence of known causes for secondary iron overload, the early diagnosis was evidenced by genotyping, revealed homozygosity for the HFE gene C282Y mutation, demonstrating the presence of hereditary hemochromatosis.

Fluorescence microscopic studies and flow cytometric measurements of lectin and hormone binding to isolated rat pancreatic acinar cells

The binding of fluorescence-labelled lectins and a fluorescence-marked hormone to the cell surface of isolated rat pancreatic acinar cells was studied by light microscopy and flow cytometric measurements. The pancreatic acinar cells were prepared by collagenase digestion. The fluorescence of cells was studied after binding of FITC-labelled WGA or UEA I as well as of FITC-marked pancreocymin/cholecystokinin (CCK-FITC) in a fluorescence microscope or FACScan. The strong binding of lectins was inhibited by preabsorption with the specific sugars. In comparison to the lectins the binding of CCK-FITC was low. There were two populations of acinar cells with different CCK-FITC binding capacity as detected by flow cytometry. The CCK-FITC cell surface fluorescence was significantly decreased by preincubation with unmarked hormone as well as with the non-labelled lectins. The inhibition of CCK-FITC binding by lectins is discussed in respect to a possible competition of the lectins and CCK for the CCK receptor.

Rapid and reliable detection of bacterial endospores in environmental samples by diagnostic electron microscopy combined with X-ray microanalysis

Diagnostic negative staining electron microscopy is a front-line method for the rapid investigation of environmental and clinical samples in emergency situations caused by bioterrorism or outbreaks of an infectious disease. Spores of anthrax are one of the diagnostic targets in case of bioterrorism, because they have been used as a bio-weapon in the past and their production and transmission are rather simple. With negative staining electron microscopy bacterial spores can be identified based on their morphology at the single cell level. However, because of their particular density, no internal structures are visible which sometimes makes it difficult to distinguish spores from particles with a similar size and shape that are frequently present in environmental samples. Spores contain a high concentration of calcium ions besides other elements, which may allow a proper discrimination of spores from other suspicious particles. To investigate this hypothesis, negative staining electron microscopy, using either transmission or scanning electron microscopes, was combined with energy dispersive X-ray microanalysis, which reveals the element content of individual nanoparticles. A peak pattern consisting of calcium, sulphur and phosphorus was found as a typical signature within the X-ray spectrum of spores in various Clostridium and Bacillus species, including all strains of anthrax (Bacillus anthracis) tested. Moreover, spores could be reliably identified by this combined approach in environmental samples, like household products, soil or various presumed bioterrorist samples. In summary, the use of X-ray spectroscopy, either directly in the transmission electron microscope, or in a correlative approach by using scanning electron microscopy, improves the emergency diagnostics of suspicious environmental samples.

Detection of gold particles in the neck skin after lightning stroke with evaporation of an ornamental chain

A German couple was struck by lightning. Both patients survived this event. Whereas the husband was unconscious for only a few minutes, his wife fell into coma for 24 h. The lightning stroke entered the body of the woman behind the left ear and left it at the left shoe. The stroke caused a partial evaporation of a gold ornamental chain on the neck, resulting in a tattoo of the neck skin. A biopsy of the skin 6 months after the event showed the accumulation of gold particles of different size in the dermis down to the subcutaneous fatty tissue. In semithin sections, histiocytes, multinucleated foreign giant cells, and fibroblasts were visible with uptaken metallic particles. In transmission electron microscopy, gold globules of up to 30 µm in diameter were visible outside the cells in the collageneous matrix of the connective tissue besides smaller metallic particles up to 5 nm inside lysosomes and residual bodies of phagocytic cells. Four different kinds of gold particles could be differentiated: globules, granular irregular particles, tubules, and tanglelike tracks. In scanning electron microscopy, gold particles were demonstrated by backscatter detection in the connective tissue of subcutis, where the EDX elemental analysis showed strong signals of aurum (Au), copper (Cu), and argentum (Ag). The detected metals were quantified by AAS as 70% gold, 21% silver, and 9% copper, which demonstrates the composition of gold alloy of the neck chain of the patient. Tanglelike tracks and elongated gold deposits represent crystals of gold salts, as detected by electron diffraction and polarization microscopy. Attempts to remove the gold particles from the skin to remove the tattoo should not be undertaken because the gold is deep and widespread.

Lectin binding studies with FITC-marked WGA and UEA I and flowcytometric measurements on isolated rat pancreatic acinar cells

Lectin binding to the glycocalyx of isolated rat pancreatic acinar cells was studied by flowcytometric measurements. The pancreatic exocrine cells were prepared after collagenase digestion and cleaned in a density gradient. The fluorescence of cells was measured in a FACScan after binding of FITC marked WGA or UEA I. The binding of lectins was inhibited by preabsorption of WGA-FITC with N-acetyl-glucosamine, sialic acid or chitinous and by preabsorption of UEA-FITC with alpha-L-fucose, respectively. Furthermore, we were able to measure a decreased WGA-FITC and UEA-FITC binding after a short preincubation of isolated cells with the peptide hormone cholecystokinin and its agonists (caerulein, pentagastrin).