Exosomes from dental pulp cells attenuate bone loss in mouse experimental periodontitis.

BACKGROUND AND OBJECTIVE: Exosomes are small vesicles secreted from many cell types. Their biological effects largely depend on their cellular origin and the physiological state of the originating cells. Exosomes secreted by mesenchymal stem cells exert therapeutic effects against multiple diseases and may serve as potential alternatives to stem cell therapies. We previously established and characterized human leukocyte antigen (HLA) haplotype homo (HHH) dental pulp cell (DPC) lines from human wisdom teeth. In this study, we aimed to investigate the effect of local administration of HHH-DPC exosomes in a mouse model of periodontitis. METHODS: Exosomes purified from HHH-DPCs were subjected to particle size analysis, and expression of exosome markers was confirmed by western blotting. We also confirmed the effect of exosomes on the migration of both HHH-DPCs and mouse osteoblastic MC3T3-E1 cells. A mouse experimental periodontitis model was used to evaluate the effect of exosomes in vivo. The morphology of alveolar bone was assessed by micro-computed tomography (µCT) and histological analysis. The effect of exosomes on osteoclastogenesis was evaluated using a co-culture system. RESULTS: The exosomes purified from HHH-DPCs were homogeneous and had a spherical membrane structure. HHH-DPC exosomes promoted the migration of both human DPCs and mouse osteoblastic cells. The MTT assay showed a positive effect on the proliferation of human DPCs, but not on mouse osteoblastic cells. Treatment with HHH-DPC exosomes did not alter the differentiation of osteoblastic cells. Imaging with µCT revealed that the exosomes suppressed alveolar bone resorption in the mouse model of periodontitis. Although no change was apparent in the dominance of TRAP-positive osteoclast-like cells in decalcified tissue sections upon exosome treatment, HHH-DPC exosomes significantly suppressed osteoclast formation in vitro. CONCLUSIONS: HHH-DPC exosomes stimulated the migration of human DPCs and mouse osteoblastic cells and effectively attenuated bone loss due to periodontitis.

Vacuolated cardiomyocytes in human endomyocardial biopsy specimens.

We encountered an unfamiliar finding during electron microscopic examination of an endomyocardial biopsy obtained from a 55-year-old woman suffering from heart failure due to dilated phase hypertrophic cardiomyopathy. Many cardiomyocytes contained large vacuoles that were mainly empty except for small amounts of amorphous substrate. These were not autophagic vacuoles, as they lacked limiting membranes. Six years later, we encountered similar histological findings in three successive biopsies sourced from another hospital. They were obtained from a 77-year-old man with hypertrophic cardiomyopathy, a 28-year-old woman with endocardial fibrosis, and a 33-year-old man with dilated cardiomyopathy. This biopsy was the second for the endocardial fibrosis patient, and her first biopsy showed no vacuoles within cardiomyocytes. Close inspection of the procedures revealed that in all of these cases the fixed biopsy specimens were carried to the hospital from other institutes using a refrigerated courier service. We then fixed rat heart tissues, froze them once, and processed them for electron microscopy. In that experiment, we were able to reproduce the vacuolar cardiomyocytes, thereby demonstrating it to be a laboratory artifact. We therefore want to emphasize to physicians not to freeze biopsy specimens and not to use a refrigerated courier service for their transport. .

Neutrophil Elastase Inhibition Ameliorates Endotoxin-induced Myocardial Injury Accompanying Degradation of Cardiac Capillary Glycocalyx.

Myocardial injury in sepsis may be caused by a burst of several inflammatory mediators, leading to vascular endothelial injuries. However, the contribution of neutrophil elastase (NE) to myocardial injury in sepsis is still unknown. We aimed to evaluate whether endotoxemia-induced myocardial injury is associated with NE. Lipopolysaccharide (LPS) was injected intraperitoneally at a dose of 20 mg/kg into granulocyte-colony-stimulating-factor knockout mice (G-CSF-KO), which have few neutrophils, and littermate control mice. The survival rate of G-CSF-KO mice 48 hours after LPS injection was significantly greater than that of control mice. The serum level of troponin I in G-CSF-KO mice was significantly lower than that in control mice. In addition, the concentration of inflammatory cytokine interleukin-6 (IL-6) was significantly decreased 6 and 12 hours after LPS administration compared with that in control mice. Ultrastructural analysis revealed that vascular endothelial structures and the endothelial glycocalyx in G-CSF-KO mice were clearly preserved. Next, mice were injected with 0.2 mg/kg sivelestat (an NE inhibitor) after LPS administration. The survival rate was significantly higher and the serum level of troponin I was lower in sivelestat-injected mice than in control mice, respectively. Furthermore, IL-6 levels were significantly decreased 6 and 12 hours after LPS administration compared with those in control mice. Vascular endothelial structures and the endothelial glycocalyx in sivelestat-treated mice were clearly preserved at the ultrastructural level. In conclusion, NE is significantly associated with myocardial injury in endotoxemia. Inhibition of NE may be a useful tool for the management of endotoxemia.

Brain-Specific Ultrastructure of Capillary Endothelial Glycocalyx and Its Possible Contribution for Blood Brain Barrier.

Endothelial glycocalyx coats healthy vascular endothelium and plays an important role in vascular homeostasis. Although cerebral capillaries are categorized as continuous, as are those in the heart and lung, they likely have specific features related to their function in the blood brain barrier. To test that idea, brains, hearts and lungs from C57BL6 mice were processed with lanthanum-containing alkaline fixative, which preserves the structure of glycocalyx, and examined using scanning and transmission electron microscopy. We found that endothelial glycocalyx is present over the entire luminal surface of cerebral capillaries. The percent area physically covered by glycocalyx within the lumen of cerebral capillaries was 40.1 ± 4.5%, which is significantly more than in cardiac and pulmonary capillaries (15.1 ± 3.7% and 3.7 ± 0.3%, respectively). Upon lipopolysaccharide-induced vascular injury, the endothelial glycocalyx was reduced within cerebral capillaries, but substantial amounts remained. By contrast, cardiac and pulmonary capillaries became nearly devoid of glycocalyx. These findings suggest the denser structure of glycocalyx in the brain is associated with endothelial protection and may be an important component of the blood brain barrier.

Ultrastructural Alteration of Pulmonary Capillary Endothelial Glycocalyx During Endotoxemia.

BACKGROUND: The most recent diagnostic criteria for sepsis include organ failure. Microvascular endothelial injury is believed to lead to the multiple organ failure seen in sepsis, although the precise mechanism is still controversial. ARDS is the primary complication during the sequential development of multiple organ dysfunction in sepsis, and endothelial injury is deeply involved. Sugar-protein glycocalyx coats all healthy vascular endothelium, and its disruption is one factor believed to contribute to microvascular endothelial dysfunction during sepsis. The goal of this study was to observe the three-dimensional ultrastructural alterations in the pulmonary capillary endothelium, including the glycocalyx, during sepsis-induced pulmonary vasculitis. METHODS: This study investigated the three-dimensional ultrastructure of pulmonary vascular endothelial glycocalyx in a mouse lipopolysaccharide-induced endotoxemia model. Lungs were fixed with lanthanum-containing alkaline fixative to preserve the glycocalyx. RESULTS: On both scanning and transmission electron microscopic imaging, the capillary endothelial glycocalyx appeared as a moss-like structure entirely covering the endothelial cell surface in normal mice. In the septic lung following liposaccharide injection, however, this structure was severely disrupted; it appeared to be peeling away and coagulated. In addition, syndecan-1 levels were significantly reduced in the septic lung, and numerous spherical structures containing glycocalyx were observed on the endothelial surface. CONCLUSIONS: It appears that endothelial glycocalyx in the lung is markedly disrupted under experimental endotoxemia conditions. This finding supports the notion that disruption of the glycocalyx is causally related to the microvascular endothelial dysfunction that is characteristic of sepsis-induced ARDS.

Three-dimensional ultrastructure of capillary endothelial glycocalyx under normal and experimental endotoxemic conditions.

BACKGROUND: Sugar-protein glycocalyx coats healthy endothelium, but its ultrastructure is not well described. Our aim was to determine the three-dimensional ultrastructure of capillary endothelial glycocalyx in the heart, kidney, and liver, where capillaries are, respectively, continuous, fenestrated, and sinusoidal. METHODS: Tissue samples were processed with lanthanum-containing alkaline fixative, which preserves the structure of glycocalyx. RESULTS: Scanning and transmission electron microscopy revealed that the endothelial glycocalyx layer in continuous and fenestrated capillaries was substantially thicker than in sinusoids. In the heart, the endothelial glycocalyx presented as moss- or broccoli-like and covered the entire luminal endothelial cell surface. In the kidney, the glycocalyx appeared to nearly occlude the endothelial pores of the fenestrated capillaries and was also present on the surface of the renal podocytes. In sinusoids of the liver, glycocalyx covered not only the luminal side but also the opposite side, facing the space of Disse. In a mouse lipopolysaccharide-induced experimental endotoxemia model, the capillary endothelial glycocalyx was severely disrupted; that is, it appeared to be peeling off the cells and clumping. Serum concentrations of syndecan-1, a marker of glycocalyx damage, were significantly increased 24 h after administration of lipopolysaccharide. CONCLUSIONS: In the present study, we visualized the three-dimensional ultrastructure of endothelial glycocalyx in healthy continuous, fenestrated, and sinusoidal capillaries, and we also showed their disruption under experimental endotoxemic conditions. The latter may provide a morphological basis for the microvascular endothelial dysfunction associated with septic injury to organs.

Ultrastructural aspects of vacuolar degeneration of cardiomyocytes in human endomyocardial biopsies.

Vacuolar degeneration of cardiomyocytes is a histological finding commonly encountered during routine light microscopic examination of human endomyocardial biopsy specimens. The vacuoles appear as intracellular clear areas lacking myofibers. By itself, this finding has little diagnostic value, but may have important clinical implications when the vacuolar contents are of etiological significance (e.g., accumulation of abnormal metabolites), and the clinical importance is increased when the disease is treatable. Thanks to its great resolving power, electron microscopy can often reveal the contents of the vacuoles and lead to a correct diagnosis. It can be used to differentially diagnose lysosomal storage diseases such as Fabry, Danon, and Pompe disease, doxorubicin cardiomyopathy, mitochondrial cardiomyopathy, autophagic degeneration, and accumulation of subcellular organelles (mitochondria, lipofuscin, glycogen granules, endoplasmic reticulum, etc.) as a nonspecific finding in failing cardiomyocytes. Nonetheless, undiagnosed cases certainly remain. It is strongly recommended that small pieces of tissue samples be fixed for electron microscopy at every endomyocardial biopsy procedure, and electron microscopic examination should be performed when a marked vacuolar degeneration is found.

Examination of composite resins with electron microscopy, microhardness tester and energy dispersive X-ray microanalyzer.

This study was conducted to examine the ultrastructures of eight recently improved light-cure restorative composite resins with scanning and transmission electron microscopes (SEM and TEM). Additionally, Vickers hardness, volume/weight fraction of filler, and chemical composition were analyzed. Composite resins selected for evaluation were Beautifil II, Clearfil AP-X, Clearfil Majesty, Estelite sigma, Filtek Supreme, Filtek Z250, Solare, and Synergy. SEM and TEM images revealed a great diversity in ultrastructure, and Vickers hardness test showed significant differences amongst all the composite resins (except between Clearfil Majesty and Estelite sigma, and between Filtek Supreme and Filtek Z250). By means of EDX, similar elements such as C, O, and Si were detected, but the concentration was different in every composite resin. Results obtained in this study served to validate that the methods employed in this study SEM and TEM at high magnification--were useful in examining the ultrastructures of composite resins. It was also found that the ultrastructure, size of filler particles, volume/weight fraction of filler, and chemical composition of the composite resins had an effect on Vickers hardness. Given the great diversity of ultrastructures amongst the composite resins, which stemmed from the different revolutionary technologies used to manufacture them, further studies are warranted in the search of clinical applications that optimally match the differing properties of these materials.

Methanogenesis versus electrogenesis: morphological and phylogenetic comparisons of microbial communities.

Two H-type microbial fuel cells were prepared. The anaerobic chambers were inoculated with rice paddy field soil and fed cellulose as an energy source. In one reactor, the anode and cathode were connected with a wire (closed circuit, CC), while they were not connected in the other reactor (open circuit, OC). The OC reactor actively produced methane. In the CC reactor, however, an electric current of 0.2 to 0.3 mA was constantly generated, and methane production was almost completely suppressed. Electron microscopy revealed that rod-shaped cells with long prosthecae-like filaments were specifically enriched in the CC reactor. Comparisons of 16S rRNA gene clone libraries revealed entirely different phylogenetic compositions in the CC and OC communities; phylotypes related to Rhizobiceae, Desulfovibrio, and Ethanoligenens were specifically enriched in the CC community. The results indicate that electrogenesis resulted in the enrichment of distinctive microbial populations and suppressed methanogenesis from cellulose.

Examination of six orthodontic adhesives with electron microscopy, hardness tester and energy dispersive X-ray microanalyzer.

OBJECTIVE: To examine the ultrastructure of six light-cure orthodontic adhesives with scanning electron microscope (SEM) and transmission electron microscope (TEM), microhardness tester, and energy dispersive X-ray microanalyzer (EDX). MATERIALS AND METHODS: The orthodontic adhesives evaluated were Transbond XT, Light Bond, BeautyOrtho Bond, Kurasper F, Heliosit Orthodontic, and Salivatect. Specimens of each adhesive were carefully prepared for observation under SEM and TEM. Furthermore, the Vickers hardness was tested, and the adhesives were evaluated with EDX. RESULTS: SEM and TEM images illustrated great diversity of the adhesives ultrastructure. The Vickers hardness test showed significant differences among all the adhesives (except Transbond XT and Salivatect). Although some similar elements were detected with EDX, the concentration was different in each adhesive. CONCLUSION: Orthodontic brackets can be bonded to the enamel surface with the adhesives available on the market. However, orthodontists might achieve better results identifying their properties and compositions.

Formation of filamentous appendages by Acinetobacter sp. Tol 5 for adhering to solid surfaces.

The toluene-degrading bacterium Acinetobacter sp. Tol 5 is highly adhesive to solid surfaces owing to two filamentous cell appendages, namely, anchors and peritrichate fibrils. When growing this bacterium in the presence of a carrier made of polyurethane foam, almost all the cells adhered to the surface of the carrier. In contrast, when Tol 5 cells were grown in the absence of the polyurethane carrier, the cells were suspended as aggregated cells or individually dispersed cells. The aggregated cells possessed the cell appendages and showed an adhesiveness similar to that of cells grown in the presence of the carrier, while the dispersed cells scarcely produced the cell appendages and showed a low level of adhesiveness. The dispersed cells started to adhere to the polyurethane carrier by producing the filamentous appendages within 30 min of the addition of the carrier as a substratum and toluene as a carbon source. Peritrichate fibrils just sprouting and growing anchors longer than 3 microm were observed when the cells started to adhere. This suggests that the presence of surface areas sufficient for adhesion might trigger cell appendage formation in Tol 5 cells for adhesion by increasing the amount of cell contact with the surfaces.

Characterization of a filamentous biofilm community established in a cellulose-fed microbial fuel cell.

BACKGROUND: Microbial fuel cells (MFCs) are devices that exploit microorganisms to generate electric power from organic matter. Despite the development of efficient MFC reactors, the microbiology of electricity generation remains to be sufficiently understood. RESULTS: A laboratory-scale two-chamber microbial fuel cell (MFC) was inoculated with rice paddy field soil and fed cellulose as the carbon and energy source. Electricity-generating microorganisms were enriched by subculturing biofilms that attached onto anode electrodes. An electric current of 0.2 mA was generated from the first enrichment culture, and ratios of the major metabolites (e.g., electric current, methane and acetate) became stable after the forth enrichment. In order to investigate the electrogenic microbial community in the anode biofilm, it was morphologically analyzed by electron microscopy, and community members were phylogenetically identified by 16S rRNA gene clone-library analyses. Electron microscopy revealed that filamentous cells and rod-shaped cells with prosthecae-like filamentous appendages were abundantly present in the biofilm. Filamentous cells and appendages were interconnected via thin filaments. The clone library analyses frequently detected phylotypes affiliated with Clostridiales, Chloroflexi, Rhizobiales and Methanobacterium. Fluorescence in-situ hybridization revealed that the Rhizobiales population represented rod-shaped cells with filamentous appendages and constituted over 30% of the total population. CONCLUSION: Bacteria affiliated with the Rhizobiales constituted the major population in the cellulose-fed MFC and exhibited unique morphology with filamentous appendages. They are considered to play important roles in the cellulose-degrading electrogenic community.

Examination of enamel-adhesive interface with focused ion beam and scanning electron microscopy.

INTRODUCTION: The purpose of this study was to observe, with a scanning electron microscope, the interface between enamel and orthodontic adhesive after focused ion-beam milling. In addition, enamel etched with phosphoric acid was compared with enamel conditioned with self-etching primer. METHODS: Four freshly extracted human premolars were collected and pumiced by using rubber cups with fluoride-free paste, washed, and dried. The enamel of 2 teeth was etched with 37% phosphoric acid for 30 seconds, washed, and dried; the enamel of the other 2 teeth was conditioned with self-etching primer for 5 seconds. Stainless steel brackets were bonded with Transbond XT adhesive (3M Unitek, Monrovia, Calif) according to the manufacturer's instructions. The specimens were milled by focused ion beam and observed under the scanning electron microscope. RESULTS: The scanning electron micrographs showed that 37% phosphoric acid seemed to produce more enamel loss than the self-etching primer. Moreover, the enamel-adhesive interface was more irregular when the enamel was etched with 37% phosphoric acid. Finally, a gentler etch pattern of the self-etching primer on the enamel surface was observed, and this conditioner could be used clinically for minimal intervention in the orthodontic bonding procedure. CONCLUSIONS: Focused ion-beam milling to prepare samples allowed clear observation of the enamel-adhesive interface without artificial damage.

Simulating the contribution of coaggregation to interspecies hydrogen fluxes in syntrophic methanogenic consortia.

A simple model (termed the syntrophy model) for simulating the contribution of coaggregation to interspecies hydrogen fluxes between syntrophic bacteria and methanogenic archaea is described. We applied it to analyzing partially aggregated syntrophic cocultures with various substrates, revealing that large fractions of hydrogen molecules were fluxed in aggregates.

Coaggregation facilitates interspecies hydrogen transfer between Pelotomaculum thermopropionicum and Methanothermobacter thermautotrophicus.

A thermophilic syntrophic bacterium, Pelotomaculum thermopropionicum strain SI, was grown in a monoculture or coculture with a hydrogenotrophic methanogen, Methanothermobacter thermautotrophicus strain DeltaH. Microscopic observation revealed that cells of each organism were dispersed in a monoculture independent of the growth substrate. In a coculture, however, these organisms coaggregated to different degrees depending on the substrate; namely, a large fraction of the cells coaggregated when they were grown on propionate, but relatively few cells coaggregated when they were grown on ethanol or 1-propanol. Field emission-scanning electron microscopy revealed that flagellum-like filaments of SI cells played a role in making contact with DeltaH cells. Microscopic observation of aggregates also showed that extracellular polymeric substance-like structures were present in intercellular spaces. In order to evaluate the importance of coaggregation for syntrophic propionate oxidation, allowable average distances between SI and DeltaH cells for accomplishing efficient interspecies hydrogen transfer were calculated by using Fick's diffusion law. The allowable distance for syntrophic propionate oxidation was estimated to be approximately 2 mum, while the allowable distances for ethanol and propanol oxidation were 16 mum and 32 mum, respectively. Considering that the mean cell-to-cell distance in the randomly dispersed culture was approximately 30 mum (at a concentration in the mid-exponential growth phase of the coculture of 5 x 10(7) cells ml(-1)), it is obvious that close physical contact of these organisms by coaggregation is indispensable for efficient syntrophic propionate oxidation.