Changes in endothelial glycocalyx layer protective ability after inflammatory stimulus.

Leukocyte adhesion to the endothelium is an important early step in the initiation and progression of sepsis. The endothelial glycocalyx layer (EGL) has been implicated in neutrophil adhesion and barrier dysfunction, but studies in this area are few. In this report, we examine the hypothesis that damage to the structure of the EGL caused by inflammation leads to increased leukocyte adhesion and endothelial barrier dysfunction. We used human umbilical vein endothelial cells enzymatically treated to remove the EGL components hyaluronic acid (HA) and heparan sulfate (HS) as a model for EGL damage. Using atomic force microscopy, we show reductions in EGL thickness after removal of either HA or HS individually, but the largest decrease, comparable with TNF-α treatment, was observed when both HA and HS were removed. Interestingly, removal of HS or HA individually did not affect neutrophil adhesion significantly, but removal of both constituents resulted in increased neutrophil adhesion. To test EGL contributions to endothelial barrier properties, we measured transendothelial electrical resistance (TEER) and diffusion of fluorescently labeled dextran (10 kDa molecular weight) across the monolayer. Removal of EGL components decreased TEER but had an insignificant effect on dextran diffusion rates. The reduction in TEER suggests that disruption of the EGL may predispose endothelial cells to increased rates of fluid leakage. These data support the view that damage to the EGL during inflammation has significant effects on the accessibility of adhesion molecules, likely facilitates leukocyte adhesion, and may also contribute to increased rates of fluid transport into tissues.

Development of Mechanical Stability in Late-Stage Embryonic Erythroid Cells: Insights From Fluorescence Imaged Micro-Deformation Studies.

The combined use of fluorescence labeling and micro-manipulation of red blood cells has proven to be a powerful tool for understanding and characterizing fundamental mechanisms underlying the mechanical behavior of cells. Here we used this approach to study the development of the membrane-associated cytoskeleton (MAS) in primary embryonic erythroid cells. Erythropoiesis comes in two forms in the mammalian embryo, primitive and definitive, characterized by intra- and extra-vascular maturation, respectively. Primitive erythroid precursors in the murine embryo first begin to circulate at embryonic day (E) 8.25 and mature as a semi-synchronous cohort before enucleating between E12.5 and E16.5. Previously, we determined that the major components of the MAS become localized to the membrane between E10.5 and E12.5, and that this localization is associated with an increase in membrane mechanical stability over this same period. The change in mechanical stability was reflected in the creation of MAS-free regions of the membrane at the tips of the projections formed when cells were aspirated into micropipettes. The tendency to form MAS-free regions decreases as primitive erythroid cells continue to mature through E14.5, at least 2 days after all detectable cytoskeletal components are localized to the membrane, indicating continued strengthening of membrane cohesion after membrane localization of cytoskeletal components. Here we demonstrate that the formation of MAS-free regions is the result of a mechanical failure within the MAS, and not the detachment of membrane bilayer from the MAS. Once a "hole" is formed in the MAS, the skeletal network contracts laterally along the aspirated projection to form the MAS-free region. In protein 4.1-null primitive erythroid cells, the tendency to form MAS-free regions is markedly enhanced. Of note, similar MAS-free regions were observed in maturing erythroid cells from human marrow, indicating that similar processes occur in definitive erythroid cells. We conclude that localization of cytoskeletal components to the cell membrane of mammalian erythroid cells during maturation is insufficient by itself to produce a mature MAS, but that subsequent processes are additionally required to strengthen intraskeletal interactions.

Micropatterned Poly(ethylene glycol) Islands Disrupt Endothelial Cell-Substrate Interactions Differently from Microporous Membranes.

Porous membranes are ubiquitous in cell co-culture and tissue-on-a-chip studies. These materials are predominantly chosen for their semi-permeable and size exclusion properties to restrict or permit transmigration and cell-cell communication. However, previous studies have shown pore size, spacing and orientation affect cell behavior including extracellular matrix production and migration. The mechanism behind this behavior is not fully understood. In this study, we fabricated micropatterned non-fouling polyethylene glycol (PEG) islands to mimic pore openings in order to decouple the effect of surface discontinuity from potential grip on the vertical contact area provided by pore wall edges. Similar to previous findings on porous membranes, we found that the PEG islands hindered fibronectin fibrillogenesis with cells on patterned substrates producing shorter fibrils. Additionally, cell migration speed over micropatterned PEG islands was greater than unpatterned controls, suggesting that disruption of cell-substrate interactions by PEG islands promoted a more dynamic and migratory behavior, similarly to enhanced cell migration on microporous membranes. Preferred cellular directionality during migration was nearly indistinguishable between substrates with identically patterned PEG islands and previously reported behavior over micropores of the same geometry, further confirming disruption of cell-substrate interactions as a common mechanism behind the cellular responses on these substrates. Interestingly, compared to respective controls, there were differences in cell spreading and a lower increase in migration speed over PEG islands compared prior results on micropores with identical feature size and spacing. This suggests that membrane pores not only disrupt cell-substrate interactions, but also provide additional physical factors that affect cellular response.

On-demand modulation of 3D-printed elastomers using programmable droplet inclusions.

One of the key thrusts in three-dimensional (3D) printing and direct writing is to seamlessly vary composition and functional properties in printed constructs. Most inks used for extrusion-based printing, however, are compositionally static and available approaches for dynamic tuning of ink composition remain few. Here, we present an approach to modulate extruded inks at the point of print, using droplet inclusions. Using a glass capillary microfluidic device as the printhead, we dispersed droplets in a polydimethylsiloxane (PDMS) continuous phase and subsequently 3D printed the resulting emulsion into a variety of structures. The mechanical characteristics of the 3D-printed constructs can be tuned in situ by varying the spatial distribution of droplets, including aqueous and liquid metal droplets. In particular, we report the use of poly(ethylene glycol) diacrylate (PEGDA) aqueous droplets for local PDMS chemistry alteration resulting in significant softening (85% reduced elastic modulus) of the 3D-printed constructs. Furthermore, we imparted magnetic functionality in PDMS by dispersing ferrofluid droplets and rationally designed and printed a rudimentary magnetically responsive soft robotic actuator as a functional demonstration of our droplet-based strategy. Our approach represents a continuing trend of adapting microfluidic technology and principles for developing the next generation of additive manufacturing technology.

Endothelial Glycocalyx Layer Properties and Its Ability to Limit Leukocyte Adhesion.

The endothelial glycocalyx layer (EGL), which consists of long proteoglycans protruding from the endothelium, acts as a regulator of inflammation by preventing leukocyte engagement with adhesion molecules on the endothelial surface. The amount of resistance to adhesive events the EGL provides is the result of two properties: EGL thickness and stiffness. To determine these, we used an atomic force microscope to indent the surfaces of cultured endothelial cells with a glass bead and evaluated two different approaches for interpreting the resulting force-indentation curves. In one, we treat the EGL as a molecular brush, and in the other, we treat it as a thin elastic layer on an elastic half-space. The latter approach proved more robust in our hands and yielded a thickness of 110 nm and a modulus of 0.025 kPa. Neither value showed significant dependence on indentation rate. The brush model indicated a larger layer thickness (∼350 nm) but tended to result in larger uncertainties in the fitted parameters. The modulus of the endothelial cell was determined to be 3.0-6.5 kPa (1.5-2.5 kPa for the brush model), with a significant increase in modulus with increasing indentation rates. For forces and leukocyte properties in the physiological range, a model of a leukocyte interacting with the endothelium predicts that the number of molecules within bonding range should decrease by an order of magnitude because of the presence of a 110-nm-thick layer and even further for a glycocalyx with larger thickness. Consistent with these predictions, neutrophil adhesion increased for endothelial cells with reduced EGL thickness because they were grown in the absence of fluid shear stress. These studies establish a framework for understanding how glycocalyx layers with different thickness and stiffness limit adhesive events under homeostatic conditions and how glycocalyx damage or removal will increase leukocyte adhesion potential during inflammation.

Robust and Gradient Thickness Porous Membranes for In Vitro Modeling of Physiological Barriers.

Porous membranes are fundamental elements for tissue-chip barrier and co-culture models. However, the exaggerated thickness of commonly available membranes may represent a stumbling block impeding a more accurate in vitro modeling. Existing techniques to fabricate membranes such as solvent cast, spin-coating, sputtering and PE-CVD result in uniform thickness films. Here, we developed a robust method to generate ultrathin porous parylene C (UPP) membranes not just with precise thicknesses down to 300 nm, but with variable gradients in thicknesses, while at the same time having porosities up to 25%. We also show surface etching and increased roughness lead to improved cell attachment. Next, we examined the mechanical properties of UPP membranes with varying porosity and thickness and fit our data to previously published models, which can help determine practical upper limits of porosity and lower limits of thickness. Lastly, we validate a straightforward approach allowing the successful integration of the UPP membranes into a prototyped 3D-printed scaffold, demonstrating mechanical robustness and allowing cell adhesion under varying flow conditions. Collectively, our results support the integration and the use of UPP membranes to examine cell-cell interaction in vitro.

How Flat Is the Tibial Osteotomy in Total Knee Arthroplasty?

BACKGROUND: Cementless total knee arthroplasty has been developed to decrease the incidence of failure in younger and more active patients. However, failures are still more common in cementless versus cemented components. It is hypothesized that this is triggered by incomplete bone-tray contact. The present study compares the final contact area of a cementless tray as a function of the initial osteotomy flatness. METHODS: Eight surgeons prepared 14 cadaveric knees for cementless total knee replacement using standard instrumentation. The topography of each osteotomy was captured with a laser scanner; 3-dimensional computer models of the surfaces were generated. After scanning each tibia, the surgeons implanted cementless tibial trays using a manual impactor. Each tibia was then dissected, embedded in mounting resin, and sectioned. The sectioned blocks were observed under stereomicroscopy to identify points of bone-tray contact which were incorporated into the 3-dimensional models. Maps were then generated illustrating depicting contacting and noncontacting areas. RESULTS: The mean initial flatness of all specimens was 1.1 ± 0.35 mm. After impaction, 79.4% ± 0.3% of the surface had established bony contact. Of the noncontacting areas, 17.6% were within 0.3 mm of the tray. Only 2.6% of the surface was at distances reported to impede ingrowth. Noncontacting areas were typically located centrally. A trend in decreasing percent contact area with increased flatness tolerance was observed (R2 = 0.605). CONCLUSION: (1) There is an inverse correlation between the flatness of the tibial osteotomy and the percentage of the bony surface in contact with underside of the tibial tray. (2) Almost all tray-tibia contact is generated during implantation through flattening of elevated features on the tibial surface. (3) Gaps between the tray and the tibia are consistently located in the central regions of the osteotomy proximal to the medullary canal.

Comparison and agreement of outcome scores through nine months after acetabular fracture fixation.

INTRODUCTION: The Modified Merle d'Aubigne-Postel Score and the Harris Hip Score are commonly used to assess the functional outcomes after acetabular fractures. A previous report showed that correlation between scores is good, that there is poor concordance among functional classes, and that the distribution of the scores is highly asymmetrical. Several issues were not addressed in this report, mainly that the data set was treated as transversal data without comparison of scores through time; therefore the objective of this article is to assess the degree of correlation and concordance between the Modified Merle d'Aubigne-Postel Score and the Harris Hip Score during the first 9 months after acetabular fracture treatment. METHODS: Both scores were recorded in a cohort of 23 previously healthy patients after 3, 6 and 9 months after fixation of acetabular fractures. Through a mixed-effects repeated measures model, we assessed differences between standardized scores. Pearson's interclass correlation coefficients for full scores and each of their domains, as well as agreement for clinical graduation classification was calculated. RESULTS: Between score correlation was 89%. We found no differences between scores at 3, 6 and 9 month follow-ups. Agreement between scores was 0.95, while agreement for clinical graduation classification was 0.67. DISCUSSION: Very short term correlation and concordance between scores is excellent, while concordance for clinical graduation classification is modest. We suggest the widespread use of the simpler score.

An Alternative Method to Characterize the Quasi-Static, Nonlinear Material Properties of Murine Articular Cartilage.

With the onset and progression of osteoarthritis (OA), articular cartilage (AC) mechanical properties are altered. These alterations can serve as an objective measure of tissue degradation. Although the mouse is a common and useful animal model for studying OA, it is extremely challenging to measure the mechanical properties of murine AC due to its small size (thickness < 50 µm). In this study, we developed novel and direct approach to independently quantify two quasi-static mechanical properties of mouse AC: the load-dependent (nonlinear) solid matrix Young's modulus (E) and drained Poisson's ratio (ν). The technique involves confocal microscope-based multiaxial strain mapping of compressed, intact murine AC followed by inverse finite element analysis (iFEA) to determine E and ν. Importantly, this approach yields estimates of E and ν that are independent of the initial guesses used for iterative optimization. As a proof of concept, mechanical properties of AC on the medial femoral condyles of wild-type mice were obtained for both trypsin-treated and control specimens. After proteolytic tissue degradation induced through trypsin treatment, a dramatic decrease in E was observed (compared to controls) at each of the three tested loading conditions. A significant decrease in ν due to trypsin digestion was also detected. These data indicate that the method developed in this study may serve as a valuable tool for comparative studies evaluating factors involved in OA pathogenesis using experimentally induced mouse OA models.

Circulating primitive erythroblasts establish a functional, protein 4.1R-dependent cytoskeletal network prior to enucleating.

Hematopoietic ontogeny is characterized by distinct primitive and definitive erythroid lineages. Definitive erythroblasts mature and enucleate extravascularly and form a unique membrane skeleton, composed of spectrin, 4.1R-complex, and ankyrinR-complex components, to survive the vicissitudes of the adult circulation. However, little is known about the formation and composition of the membrane skeleton in primitive erythroblasts, which progressively mature while circulating in the embryonic bloodstream. We found that primary primitive erythroblasts express the major membrane skeleton genes present in similarly staged definitive erythroblasts, suggesting that the composition and formation of this membrane network is conserved in maturing primitive and definitive erythroblasts despite their respective intravascular and extravascular locations. Membrane deformability and stability of primitive erythroblasts, assayed by microfluidic studies and fluorescence imaged microdeformation, respectively, significantly increase prior to enucleation. These functional changes coincide with protein 4.1 R isoform switching and protein 4.1R-null primitive erythroblasts fail to establish normal membrane stability and deformability. We conclude that maturing primitive erythroblasts initially navigate the embryonic vasculature prior to establishing a deformable cytoskeleton, which is ultimately formed prior to enucleation. Formation of an erythroid-specific, protein 4.1R-dependent membrane skeleton is an important feature not only of definitive, but also of primitive, erythropoiesis in mammals.

Stabilization of carbon dioxide (CO2) bubbles in micrometer-diameter aqueous droplets and the formation of hollow microparticles.

We report an approach to stabilize carbon dioxide (CO2) gas bubbles encapsulated in micrometer-diameter aqueous drops when water in the aqueous drops is evaporated. CO2-in-water-in-oil double emulsion drops are generated using microfluidic approaches and evaporation is conducted in the presence of sodium dodecyl sulfate (SDS), poly(vinyl alcohol) (PVA) and/or graphene oxide (GO) particles dispersed in the aqueous phase of the double emulsion drops. We examine the roles of the bubble-to-drop size ratio, PVA and GO concentration in the stabilization of CO2 bubbles upon water evaporation and show that thin-shell particles with encapsulated CO2 bubbles can be obtained under optimized conditions. The developed approach offers a new strategy to study CO2 dissolution and stability on the microscale and the synthesis of novel gas-core microparticles.

Bmi-1 Regulates Extensive Erythroid Self-Renewal.

Red blood cells (RBCs), responsible for oxygen delivery and carbon dioxide exchange, are essential for our well-being. Alternative RBC sources are needed to meet the increased demand for RBC transfusions projected to occur as our population ages. We previously have discovered that erythroblasts derived from the early mouse embryo can self-renew extensively ex vivo for many months. To better understand the mechanisms regulating extensive erythroid self-renewal, global gene expression data sets from self-renewing and differentiating erythroblasts were analyzed and revealed the differential expression of Bmi-1. Bmi-1 overexpression conferred extensive self-renewal capacity upon adult bone-marrow-derived self-renewing erythroblasts, which normally have limited proliferative potential. Importantly, Bmi-1 transduction did not interfere with the ability of extensively self-renewing erythroblasts (ESREs) to terminally mature either in vitro or in vivo. Bmi-1-induced ESREs can serve to generate in vitro models of erythroid-intrinsic disorders and ultimately may serve as a source of cultured RBCs for transfusion therapy.

CD44 variant isoforms expressed by breast cancer cells are functional E-selectin ligands under flow conditions.

Adhesion of circulating tumor cells to vascular endothelium is mediated by specialized molecules that are functional under shear forces exerted by hematogenous flow. Endothelial E-selectin binding to glycoforms of CD44 mediates shear-resistant cell adhesion in numerous physiological and pathological conditions. However, this pathway is poorly understood in breast cancer and is the focus of the present investigation. All breast cancer cell lines used in this study strongly expressed CD44. In particular, BT-20 cells expressed CD44s and multiple CD44v isoforms, whereas MDA-MB-231 cells predominantly expressed CD44s but weakly expressed CD44v isoforms. CD44 expressed by BT-20, but not MDA-MB-231, cells possessed E-selectin ligand activity as detected by Western blotting and antigen capture assays. Importantly, CD44 expressed by intact BT-20 cells were functional E-selectin ligands, regulating cell rolling and adhesion under physiological flow conditions, as found by shRNA-targeted silencing of CD44. Antigen capture assays strongly suggest greater shear-resistant E-selectin ligand activity of BT-20 cell CD44v isoforms than CD44s. Surprisingly, CD44 was not recognized by the HECA-452 MAb, which detects sialofucosylated epitopes traditionally expressed by selectin ligands, suggesting that BT-20 cells express a novel glycoform of CD44v as an E-selectin ligand. The activity of this glycoform was predominantly attributed to N-linked glycans. Furthermore, expression of CD44v as an E-selectin ligand correlated with high levels of fucosyltransferase-3 and -6 and epithelial, rather than mesenchymal, cell phenotype. Together, these data demonstrate that expression of CD44 as a functional E-selectin ligand may be important in breast cancer metastasis.

Expression of E-selectin ligands on circulating tumor cells: cross-regulation with cancer stem cell regulatory pathways?

Although significant progress has been made in the fight against cancer, successful treatment strategies have yet to be developed to combat those tumors that have metastasized to distant organs. Poor characterization of the molecular mechanisms of cancer spread is a major impediment to designing predictive diagnostics and effective clinical interventions against late stage disease. In hematogenous metastasis, it is widely suspected that circulating tumor cells (CTCs) express specific adhesion molecules that actively initiate contact with the vascular endothelium lining the vessel walls of the target organ. This "tethering" is mediated by ligands expressed by CTCs that bind to E-selectin expressed by endothelial cells. However, it is currently unknown whether expression of functional E-selectin ligands on CTCs is related to cancer stem cell regulatory or maintenance pathways, particularly epithelial-to-mesenchymal transition and the reverse, mesenchymal-to-epithelial transition. In this hypothesis and theory article, we explore the potential roles of these mechanisms on the dynamic regulation of selectin ligands mediating CTC trafficking during metastasis.

HECA-452 is a non-function blocking antibody for isolated sialyl Lewis x adhesion to endothelial expressed E-selectin under flow conditions.

E-selectin, expressed on inflamed endothelium, and sialyl Lewis x (sLe(x)), present on the surface of leukocytes, play a key role in leukocyte-endothelial interactions during leukocyte recruitment to sites of inflammation. HECA-452 is a monoclonal antibody (mAb) that recognizes sLe(x) and is routinely used by investigators from diverse fields who seek to unravel the mechanisms of leukocyte adhesion. The data regarding the ability of HECA-452 to inhibit carbohydrate-mediated leukocyte adhesion to E-selectin remains conflicted, in part due to the presence of a variety of potential E-selectin reactive moieties on leukocytes. Recognizing this, we utilized a complementary approach to gain insight into HECA-452 adhesion assays. Specifically, we used sLe(x) microspheres to investigate the hypothesis that HECA-452 is a non-function blocking mAb for isolated sLe(x) mediated adhesion to endothelial expressed E-selectin. Flow cytometric analysis revealed that HECA-452 recognizes and binds to the sLe(x) microspheres. Perfusion of the sLe(x) microspheres over human umbilical vein endothelial cells (HUVEC) at 1.5 dyn/cm² revealed that the microspheres attach to 4h interleukin (IL)-1ß activated HUVEC specifically via E-selectin. Pretreatment of the sLe(x) microspheres with HECA-452 did not influence sLe(x) microsphere initial tethering and accumulation on IL-1ß activated HUVEC. Neuraminidase and fucosidase treatments of sLe(x) microspheres revealed that sialic acid and fucose are required for E-selectin binding, whereas HECA-452 recognition of sLe(x) does not depend on the fucose moiety to the extent required for E-selectin recognition. This latter finding suggests there are potential subtle differences between the sLe(x) antigens for E-selectin and HECA-452. Combined, the data indicate that HECA-452 is a non-inhibitor of sLe(x)-mediated adhesion to endothelial expressed E-selectin.