scholarly journals Surface engineering of titania nanotubes incorporated with double-layered extracellular vesicles to modulate inflammation and osteogenesis

2021 ◽  
Vol 8 (3) ◽  
Author(s):  
Qingyu Zhao ◽  
Yi Zhang ◽  
Lan Xiao ◽  
Haiping Lu ◽  
Yaping Ma ◽  
...  
Keyword(s):  
Bone Regeneration ◽  
Extracellular Vesicles ◽  
Laser Scanning ◽  
Surface Engineering ◽  
Titanium Implant ◽  
Laser Scanning Microscopy ◽  
Titania Nanotubes ◽  
Confocal Laser ◽  
Gene And Protein Expression ◽  
Modification Technique

Abstract Titania nanotubes (TNT) generated on titanium implant are emerged as important modification technique to facilitate bone regeneration. Mesenchymal stem cells (MSCs)-derived exosomes are membrane bound extracellular vesicles (EVs), which play an important role in tissue regeneration. The objective of this study was to generate an EVs hybrid TNT aiming at regulating inflammation, MSCs recruitment and osteogenesis. We isolated EVs from MSCs (MSCs EVs) and 3-day osteogenically differentiated MSCs (3d EVs). MSC EVs and 3d EVs exhibited round morphology under TEM, which also showed robust internalization by human bone marrow derived MSCs (hBMSCs). Next, we fabricated 3d EVs/MSC EVs hybrid TNT. When inflammatory macrophages were co-cultured with EVs hybrid TNT, the gene and protein expression of inflammatory cytokine were significantly reduced. Macrophage morphology was also examined by confocal laser scanning microscopy (CLSM) and scanning electron microscopy (SEM). Further migratory ability study using hBMSCs indicated significant enhancement of MSCs migration in EVs hybrid TNT. In addition, we further demonstrated significant increase of osteogenic differentiation of hBMSCs in EVs hybrid TNT. This study suggests that EVs hybrid TNT may serve as a viable therapeutic approach to enhance osteogenesis and bone regeneration.

scholarly journals Identification of PDGFRα-positive interstitial cells in the distal segment of the murine vas deferens

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Tasuku Hiroshige ◽  
Kei-Ichiro Uemura ◽  
Shingo Hirashima ◽  
Kiyosato Hino ◽  
Akinobu Togo ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Extracellular Vesicles ◽  
Laser Scanning ◽  
Vas Deferens ◽  
Smooth Muscles ◽  
Growth Factor Receptor ◽  
Distal Segment ◽  
Interstitial Cells ◽  
Laser Scanning Microscopy ◽  
Confocal Laser

AbstractPlatelet-derived growth factor receptor-α (PDGFRα)-positive interstitial cells (ICs) are widely distributed in various organs and may be involved in the motility of various tubular organs. We, for the first time, aimed to investigate the distribution, immunohistochemical characteristics, and ultrastructure of PDGFRα-positive ICs in murine vas deferens, using confocal laser scanning microscopy, transmission electron microscopy (TEM), and immuno-electron microscopy (immuno-EM). For immunofluorescence, we used antibodies against PDGFRα and other markers of ICs. PDGFRα-positive ICs were distributed widely in the lamina propria, smooth muscles, and serosal layers. Although most PDGFRα-positive ICs labeled CD34, they did not label CD34 in the subepithelial layers. Additionally, PDGFRα-positive ICs were in close proximity to each other, as also to the surrounding cells. TEM and immuno-EM findings revealed that PDGFRα-positive ICs established close physical interactions with adjacent ICs. Extracellular vesicles were also detected around the PDGFRα-positive ICs. Our morphological findings suggest that PDGFRα-positive ICs may have several subpopulations, which can play an important role in intercellular signaling via direct contact with the IC network and the extracellular vesicles in the murine vas deferens. Further investigation on PDGFRα-positive ICs in the vas deferens may lead to understanding the vas deferens mortility.

scholarly journals Translational Research for Orthopedic Bone Graft Development

Materials ◽  
2021 ◽  
Vol 14 (15) ◽  
pp. 4130
Author(s):  
Maria J. C. Vilela ◽  
Bruno J. A. Colaço ◽  
José Ventura ◽  
Fernando J. M. Monteiro ◽  
Christiane L. Salgado
Keyword(s):  
Production Process ◽  
Bone Regeneration ◽  
Osteogenic Differentiation ◽  
Laser Scanning ◽  
Complex Structure ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Mechanical Resistance ◽  
Human Mscs ◽  
Biological Studies

Designing biomaterials for bone-substitute applications is still a challenge regarding the natural complex structure of hard tissues. Aiming at bone regeneration applications, scaffolds based on natural collagen and synthetic nanohydroxyapatite were developed, and they showed adequate mechanical and biological properties. The objective of this work was to perform and evaluate a scaled-up production process of this porous biocomposite scaffold, which promotes bone regeneration and works as a barrier for both fibrosis and the proliferation of scar tissue. The material was produced using a prototype bioreactor at an industrial scale, instead of laboratory production at the bench, in order to produce an appropriate medical device for the orthopedic market. Prototypes were produced in porous membranes that were e-beam irradiated (the sterilization process) and then analysed by scanning electron microscopy (SEM), confocal laser scanning microscopy (CLSM), dynamic mechanical analysis (DMA), cytotoxicity tests with mice fibroblasts (L929), human osteoblast-like cells (MG63) and human MSC osteogenic differentiation (HBMSC) with alkaline phosphatase (ALP) activity and qPCR for osteogenic gene expression. The prototypes were also implanted into critical-size bone defects (rabbits’ tibia) for 5 and 15 weeks, and after that were analysed by microCT and histology. The tests performed for the physical characterization of the materials showed the ability of the scaffolds to absorb and retain water-based solvents, as well as adequate mechanical resistance and viscoelastic properties. The cryogels had a heteroporous morphology with microporosity and macroporosity, which are essential conditions for the interaction between the cells and materials, and which consequently promote bone regeneration. Regarding the biological studies, all of the studied cryogels were non-cytotoxic by direct or indirect contact with cells. In fact, the scaffolds promoted the proliferation of the human MSCs, as well as the expression of the osteoblastic phenotype (osteogenic differentiation). The in vivo results showed bone tissue ingrowth and the materials’ degradation, filling the critical bone defect after 15 weeks. Before and after irradiation, the studied scaffolds showed similar properties when compared to the results published in the literature. In conclusion, the material production process upscaling was optimized and the obtained prototypes showed reproducible properties relative to the bench development, and should be able to be commercialized. Therefore, it was a successful effort to harness knowledge from the basic sciences to produce a new biomedical device and enhance human health and wellbeing.

scholarly journals Characterization of hyaluronan-coated extracellular vesicles in synovial fluid of patients with osteoarthritis and rheumatoid arthritis

2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Anne-Mari Mustonen ◽  
Janne Capra ◽  
Kirsi Rilla ◽  
Petri Lehenkari ◽  
Sanna Oikari ◽  
...  
Keyword(s):  
Rheumatoid Arthritis ◽  
Synovial Fluid ◽  
Extracellular Vesicles ◽  
Laser Scanning ◽  
Significant Proportion ◽  
Study Groups ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Replacement Surgery ◽  
Human Knee Joint

Abstract Background Hyaluronic acid (HA) is the major extracellular matrix glycosaminoglycan with a reduced synovial fluid (SF) concentration in arthropathies. Cell-derived extracellular vesicles (EV) have also been proposed to contribute to pathogenesis in joint diseases. It has recently been shown that human SF contains HA-coated EV (HA–EV), but their concentration and function in joint pathologies remain unknown. Methods The aim of the present study was to develop an applicable method based on confocal laser scanning microscopy (CLSM) and image analysis for the quantification of EV, HA-particles, and HA–EV in the SF of the human knee joint. Samples were collected during total knee replacement surgery from patients with end-stage rheumatoid arthritis (RA, n = 8) and osteoarthritis (OA, n = 8), or during diagnostic/therapeutic arthroscopy unrelated to OA/RA (control, n = 7). To characterize and quantify EV, HA-particles, and HA–EV, SF was double-stained with plasma membrane and HA probes and visualized by CLSM. Comparisons between the patient groups were performed with the Kruskal–Wallis analysis of variance. Results The size distribution of EV and HA-particles was mostly similar in the study groups. Approximately 66% of EV fluorescence was co-localized with HA verifying that a significant proportion of EV carry HA. The study groups were clearly separated by the discriminant analysis based on the CLSM data. The intensities of EV and HA-particle fluorescences were lower in the RA than in the control and OA groups. Conclusions CLSM analysis offers a useful tool to assess HA–EV in SF samples. The altered EV and HA intensities in the RA SF could have possible implications for diagnostics and therapy.

3-D imaging of cells using a confocal laser scanning microscope and digital image processing

1988 ◽  
Vol 46 ◽  
pp. 96-97
Author(s):  
Thomas M. Jovin ◽  
Michel Robert-Nicoud ◽  
Donna J. Arndt-Jovin ◽  
Thorsten Schormann
Keyword(s):  
Laser Scanning ◽  
Confocal Laser Scanning Microscope ◽  
Laser Scanning Microscopy ◽  
Confocal Laser Scanning ◽  
Confocal Laser ◽  
Scanning Microscope ◽  
Laser Scanning Microscope ◽  
Biochemical Processes ◽  
Adjacent Structures

Light microscopic techniques for visualizing biomolecules and biochemical processes in situ have become indispensable in studies concerning the structural organization of supramolecular assemblies in cells and of processes during the cell cycle, transformation, differentiation, and development. Confocal laser scanning microscopy offers a number of advantages for the in situ localization and quantitation of fluorescence labeled targets and probes: (i) rejection of interfering signals emanating from out-of-focus and adjacent structures, allowing the “optical sectioning” of the specimen and 3-D reconstruction without time consuming deconvolution; (ii) increased spatial resolution; (iii) electronic control of contrast and magnification; (iv) simultanous imaging of the specimen by optical phenomena based on incident, scattered, emitted, and transmitted light; and (v) simultanous use of different fluorescent probes and types of detectors.We currently use a confocal laser scanning microscope CLSM (Zeiss, Oberkochen) equipped with 3-laser excitation (u.v - visible) and confocal optics in the fluorescence mode, as well as a computer-controlled X-Y-Z scanning stage with 0.1 μ resolution.

Vacuoles Induced in Mammalian Cells by Helicobacter Cytotoxin are Acidic Organelles

1990 ◽  
Vol 48 (3) ◽  
pp. 168-169
Author(s):  
M. H. Chestnut ◽  
C. E. Catrenich
Keyword(s):  
Acridine Orange ◽  
Mammalian Cells ◽  
Laser Scanning ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Ulcer Disease ◽  
Gastroduodenal Disease ◽  
H Pylori

Helicobacter pylori is a non-invasive, Gram-negative spiral bacterium first identified in 1983, and subsequently implicated in the pathogenesis of gastroduodenal disease including gastritis and peptic ulcer disease. Cytotoxic activity, manifested by intracytoplasmic vacuolation of mammalian cells in vitro, was identified in 55% of H. pylori strains examined. The vacuoles increase in number and size during extended incubation, resulting in vacuolar and cellular degeneration after 24 h to 48 h. Vacuolation of gastric epithelial cells is also observed in vivo during infection by H. pylori. A high molecular weight, heat labile protein is believed to be responsible for vacuolation and to significantly contribute to the development of gastroduodenal disease in humans. The mechanism by which the cytotoxin exerts its effect is unknown, as is the intracellular origin of the vacuolar membrane and contents. Acridine orange is a membrane-permeant weak base that initially accumulates in low-pH compartments. We have used acridine orange accumulation in conjunction with confocal laser scanning microscopy of toxin-treated cells to begin probing the nature and origin of these vacuoles.

Use of confocal laser scanning microscopy and a computer model to understand ink cavitation and filamentation

TAPPI Journal ◽  
2010 ◽  
Vol 9 (10) ◽  
pp. 7-15
Author(s):  
HANNA KOIVULA ◽  
DOUGLAS BOUSFIELD ◽  
MARTTI TOIVAKKA
Keyword(s):  
Laser Scanning ◽  
Confocal Laser Scanning Microscope ◽  
Laser Scanning Microscopy ◽  
Confocal Laser Scanning ◽  
Confocal Laser ◽  
Print Quality ◽  
Length Scale ◽  
Offset Printing ◽  
Significant Difference ◽  
Printing Speed

In the offset printing process, ink film splitting has an important impact on formation of ink filaments. The filament size and its distribution influence the leveling of ink and hence affect ink setting and the print quality. However, ink filaments are difficult to image due to their short lifetime and fine length scale. Due to this difficulty, limited work has been reported on the parameters that influence filament size and methods to characterize it. We imaged ink filament remains and quantified some of their characteristics by changing printing speed, ink amount, and fountain solution type. Printed samples were prepared using a laboratory printability tester with varying ink levels and operating settings. Rhodamine B dye was incorporated into fountain solutions to aid in the detection of the filaments. The prints were then imaged with a confocal laser scanning microscope (CLSM) and images were further analyzed for their surface topography. Modeling of the pressure pulses in the printing nip was included to better understand the mechanism of filament formation and the origin of filament length scale. Printing speed and ink amount changed the size distribution of the observed filament remains. There was no significant difference between fountain solutions with or without isopropyl alcohol on the observed patterns of the filament remains.

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