Biochemical and Structural Characterization of Neocartilage Formed by Mesenchymal Stem Cells in Alginate Hydrogels

A popular approach to make neocartilage in vitro is to immobilize cells with chondrogenic potential in hydrogels. However, functional cartilage cannot be obtained by control of cells only, as function of cartilage is largely dictated by architecture of extracellular matrix (ECM). Therefore, characterization of the cells, coupled with structural and biochemical characterization of ECM, is essential in understanding neocartilage assembly to create functional implants in vitro. We focused on mesenchymal stem cells (MSC) immobilized in alginate hydrogels, and used immunohistochemistry (IHC) and gene expression analysis combined with advanced microscopy techniques to describe properties of cells and distribution and organization of the forming ECM. In particular, we used second harmonic generation (SHG) microscopy and focused ion beam/scanning electron microscopy (FIB/SEM) to study distribution and assembly of collagen. Samples with low cell seeding density (1e7 MSC/ml) showed type II collagen molecules distributed evenly through the hydrogel. However, SHG microscopy clearly indicated only pericellular localization of assembled fibrils. Their distribution was improved in hydrogels seeded with 5e7 MSC/ml. In those samples, FIB/SEM with nm resolution was used to visualize distribution of collagen fibrils in a three dimensional network extending from the pericellular region into the ECM. In addition, distribution of enzymes involved in procollagen processing were investigated in the alginate hydrogel by IHC. It was discovered that, at high cell seeding density, procollagen processing and fibril assembly was also occurring far away from the cell surface, indicating sufficient transport of procollagen and enzymes in the intercellular space. At lower cell seeding density, the concentration of enzymes involved in procollagen processing was presumably too low. FIB/SEM and SHG microscopy combined with IHC localization of specific proteins were shown to provide meaningful insight into ECM assembly of neocartilage, which will lead to better understanding of cartilage formation and development of new tissue engineering strategies

Seeing a Mycobacterium-Infected Cell in Nanoscale 3D: Correlative Imaging by Light Microscopy and FIB/SEM Tomography

<div><p>Mycobacteria pose a threat to the world health today, with pathogenic and opportunistic bacteria causing tuberculosis and non-tuberculous disease in large parts of the population. Much is still unknown about the interplay between bacteria and host during infection and disease, and more research is needed to meet the challenge of drug resistance and inefficient vaccines. This work establishes a reliable and reproducible method for performing correlative imaging of human macrophages infected with mycobacteria at an ultra-high resolution and in 3D. Focused Ion Beam/Scanning Electron Microscopy (FIB/SEM) tomography is applied, together with confocal fluorescence microscopy for localization of appropriately infected cells. The method is based on an Aclar poly(chloro-tri-fluoro)ethylene substrate, micropatterned into an advantageous geometry by a simple thermomoulding process. The platform increases the throughput and quality of FIB/SEM tomography analyses, and was successfully applied to detail the intracellular environment of a whole mycobacterium-infected macrophage in 3D.</p></div

Master design and aclar substrate production.

<p>A silicon master was designed and produced in order to pattern aclar films by thermomoulding. a) The master design, including an array of 100 μm sized squares and a reference system for simple localization of cells with various imaging techniques. b) Micropatterned aclar substrates were produced from the master by thermomoulding. The master was heated to 230°C, before a piece of aclar and a glass microscopy slide was placed on top. An even pressure was applied to the assembly, before subsequently cooling down and separating the individual components. After thermomoulding, the microblocks from the master became wells in the aclar substrate. c) Illustration of how primary human macrophages could be cultured in the wells. After <i>M</i>. <i>avium</i> infection and confocal imaging, cells were fixed, stained, dehydrated and embedded in epoxy for FIB/SEM tomography. The aclar substrate was removed after resin polymerization, creating an array of protruding microblocks containing immobilized cells.</p

Volume correlation procedure.

<p>One entire microwell was imaged by light microscopy (DIC and confocal fluorescence microscopy), while one cell at an edge of the same well was imaged by FIB/SEM tomography. a) Overview SEM image of the well of interest after epoxy embedding, and SEM image of the region of interest at the corner of the well after preparing the region for FIB/SEM tomography. The images collected during the tomography experiment are in the x-z plane (yellow square), while the images collected during LM imaging are in the x-y plane, parallel to the well surface (yellow line). Ion images are collected in the x-y plane before each milling step, where the cross serves as a reference marker enabling each slice to be milled with nanometer precision. b) Illustration of how the shape of the microwells can be used to place the volume imaged during FIB/SEM tomography correctly within the larger volume imaged during LM imaging. The ion images are first aligned with the edges of the well as imaged by LM. The perpendicular FIB/SEM tomography images can then be aligned with the ion images, with the first and the last image overlapping with the edge observed in the first and last ion image. c) After alignment the two volumes are correlated, and an overlay between fluorescence and EM images can be done in any plane or volume. In the 2D overlay, an xy projection from the FIB/SEM stack is overlaid on the corresponding fluorescence image, with nuclei displayed in blue and bacteria in red. Scale bars: a: 20μm, c: 5μm.</p

3D reconstruction after correlative imaging.

<p>a) and b) 3D models of the same cell, reconstructed from a FIB/SEM tomography stack and a confocal z stack respectively. The nuclei are displayed in blue and mycobacteria in red. The correspondence between the two models is good.</p

FIB/SEM imaging.

<p>After confocal imaging, <i>M</i>. <i>avium</i>-infected primary human macrophages were dehydrated, stained and embedded in epoxy. a) SEM micrograph of the final epoxy sample surface after removal of the aclar substrate. The topography of the reference system is sufficient to remain visible at this level. All cells located at an edge of a small protruding block are immediately accessible for FIB/SEM tomography analyses. b) After localization of a region of interest (indicated by the black rectangle in a), the sample was tilted to 52° and the cell of interest was exposed by ion-beam milling. c). SEM micrograph collected during a FIB/SEM tomography experiment, from the surface area indicated by a white rectangle in b). White arrows indicate the following observable details: 1: mycobacteria surrounded by phagosomal membranes, 2: Golgi, 3: nuclear pores recognized by discontinuities in the double nuclear membrane, 4: mitochondrion. Scale bars: 10 μm in b, 2μm in c. The blocks in a) are about 100μm wide.</p

Light microscopy.

<p>Primary human macrophages were grown and infected with CFP-expressing <i>M</i>. <i>avium</i> on aclar microwell substrates. The cells were then aldehyde fixed and fluorescence stained for nucleus, before light microscopy imaging was performed. a) DIC image illustrating the excellent visibility of both microwells and reference system that were imprinted in aclar by thermomoulding. b) Confocal fluorescence microscopy image with DIC overlay, displaying nuclei in blue and bacteria in red. About 70% of infected macrophages contained at least one bacterium (n = 100). Scale bar 200μm.</p

3D models.

<p>Selected structures were reconstructed in 3D from the image stack obtained during a FIB/SEM tomography experiment. a) Column 1 shows a representative SEM image from the original stack while column two outlines how the segmentation was performed on these images to create the 3D models shown in column 3. Nuclei are colored in blue, mitochondria in green, a continuous vesicular structure in yellow, bacteria in red and a selected phagosome in cyan. In row 4, white arrows point to two phagosomal membranes surrounding. Similar membranes were traced to reconstruct the phagosome enveloping 3 bacteria in the lower right Fig b) 3D model including nuclei, vesicle and bacteria. Scale bars: Nucleus and vesicle: 5μm, Mitochondria and bacteria/phagosomes: 3μm.</p

Plasma membrane damage causes NLRP3 activation and pyroptosis during Mycobacterium tuberculosis infection

Mycobacterium tuberculosis is a global health problem in part as a result of extensive cytotoxicity caused by the infection. Here, we show how M. tuberculosis causes caspase-1/NLRP3/gasdermin D-mediated pyroptosis of human monocytes and macrophages. A type VII secretion system (ESX-1) mediated, contact-induced plasma membrane damage response occurs during phagocytosis of bacteria. Alternatively, this can occur from the cytosolic side of the plasma membrane after phagosomal rupture in infected macrophages. This damage causes K+ efflux and activation of NLRP3-dependent IL-1β release and pyroptosis, facilitating the spread of bacteria to neighbouring cells. A dynamic interplay of pyroptosis with ESCRT-mediated plasma membrane repair also occurs. This dual plasma membrane damage seems to be a common mechanism for NLRP3 activators that function through lysosomal damage