Effective orifice diameter: a new sizing parameter of surgical valve prostheses to inform valve selection.

Background: The labeled sizes of surgical valve prostheses and their discordance with the physical internal valve orifice sizes has long been a controversy in the cardiac surgery community, leading many to believe it to be a contributing factor in prosthesis-patient mismatch following valvular replacement surgery. In an attempt to address this issue, the International Organization for Standardization (ISO) 5840-2:2021 standard for surgical valve prostheses recommends that a new sizing parameter, namely, the effective orifice diameter, be provided in labeling by all manufacturers as an indicator of the true flow-passing capacity of a prosthetic valve. Methods: The ISO Cardiac Valves Working Group conducted a multi-laboratory round-robin study to investigate whether the effective orifice diameter of a prosthetic surgical valve could be derived repeatably and reproducibly through steady forward-flow testing. A total of seven valve models, each with multiple sizes, were tested, including a mechanical heart valve and multiple biological heart valves. Results: The round-robin study confirmed that the steady forward-flow test had good intra-laboratory repeatability and inter-laboratory reproducibility in deriving the effective orifice diameters of surgical valve prostheses. On average, among the participating laboratories, the experimentally derived effective orifice diameter of a prosthetic heart valve was 3-12 mm smaller than its labeled size. Conclusions: The effective orifice diameter provides better characterization of the hydrodynamic characteristics of a surgical valve prosthesis and can be derived using a validated steady forward-flow test method. This new sizing parameter will soon be adopted by surgical valve manufacturers and provided in device labeling to inform valve selection by surgeons.

A simple 3-point flexural method for measuring fracture toughness of the dental porcelain to zirconia bond and other brittle bimaterial interfaces.

PURPOSE: Porcelain fused to zirconia prostheses are widely used, but porcelain chipping, fracture, spalling and delamination are common clinical problems. Conventional bond strength testing is inherently unsuited for studying interfacial failure by cracking in brittle materials. Instead, fracture toughness is a more meaningful parameter because it can assess the robustness of the interface when subjected to loading, but fracture mechanics approaches have only rarely been used. Our purpose was to develop a novel, simple, 3-point flexural methodology and mathematical analysis to measure the fracture toughness of the porcelain to zirconia interface. METHODS: Equations were derived to estimate the fracture toughness of the bond by computing the interfacial energy release rate for a novel simple 3-point flexural test model. The test was validated using two different configurations of layered zirconia/porcelain beams (n = 10), approximating the dimensions of a fixed dental prosthesis, fabricated from a tetragonal polycrystalline zirconium dioxide partially stabilized with yttria and a feldspathic dental porcelain. RESULTS: Cracking along the bimaterial interface was produced and measured as a discrete event. Fracture toughness means (standard deviations) computed from the measured energy release rate, for the porcelain to zirconia interface in two different specimen configurations were 7.9 (1.3) and 5.3 (1.6) J/m2. CONCLUSIONS: Equations were derived to measure interfacial fracture toughness of brittle materials using a novel simple 3-point flexural test method. The test was then validated; estimates for the fracture toughness for the porcelain to zirconia bond, overlapped with previously published data derived from more complex 4-point notched tests.

Effect of tooth substrate and porcelain thickness on porcelain veneer failure loads in vitro.

STATEMENT OF PROBLEM: Bonded porcelain veneers are widely used esthetic restorations. High success and survival rates have been reported, but failures do occur. Fractures are the commonest failure mode. Minimally invasive or thin veneers have gained popularity. Increased enamel and porcelain thickness improve the strength of veneers bonded to enamel, but less is known about dentin or mixed substrates. PURPOSE: The purpose of this in vitro study was to measure the influences of tooth substrate type (all-enamel, all-dentin, or half-dentin-half-enamel) and veneer thickness on the loads needed to cause initial and catastrophic porcelain veneer failure. MATERIAL AND METHODS: Model discoid porcelain veneer specimens of varying thicknesses were bonded to the flattened facial surfaces of incisors with different enamel and dentin tooth substrates, artificially aged, and loaded to failure with a small sphere. Initial and catastrophic fracture events were identified and analyzed statistically and fractographically. RESULTS: Fracture events included initial Hertzian cracks, intermediate radial cracks, and catastrophic gross failure. All specimens retained some porcelain after catastrophic failure. Cement failure occurred at the cement-porcelain interface not at the cement-tooth interface. Porcelain veneers bonded to enamel were substantially stronger and more damage-tolerant than those bonded to dentin or mixed substrates. Increased porcelain thickness substantially raised the loads to catastrophic failure on enamel substrates but only moderately raised the loads to catastrophic failure on dentin or mixed substrates. The veneers bonded to half-dentin-half-enamel behaved remarkably like those bonded wholly to dentin. CONCLUSIONS: Porcelain veneers bonded to enamel were substantially stronger and more damage-tolerant than those bonded to dentin or half-enamel-half dentin.

Effect of porcelain and enamel thickness on porcelain veneer failure loads in vitro.

STATEMENT OF PROBLEM: Bonded porcelain veneers are widely used esthetic restorations. Although high success and survival rates have been reported, failures occur. Fracture is the most common failure mode. Fractures range from incomplete cracks to the catastrophic. Minimally invasive or thin partial veneers have gained popularity. PURPOSE: The aim of this study was to measure the influences of porcelain veneer thickness and enamel substrate thickness on the loads needed to cause the initial fracture and catastrophic failure of porcelain veneers. MATERIAL AND METHODS: Model discoid porcelain veneer specimens of varying thickness were bonded to the flattened facial surfaces of incisors, artificially aged, and loaded to failure with a small sphere. Individual fracture events were identified and analyzed statistically and fractographically. RESULTS: Fracture events included initial Hertzian cracks, intermediate radial cracks, and catastrophic gross failure. Increased porcelain, enamel, and their combined thickness had like effects in substantially raising resistance to catastrophic failure but also slightly decreased resistance to initial Hertzian cracking. Fractographic and numerical data demonstrated that porcelain and tooth enamel behaved in a remarkably similar manner. As porcelain thickness, enamel thickness, and their combined thickness increased, the loads needed to produce initial fracture and catastrophic failure rose substantially. Porcelain veneers withstood considerable damage before catastrophic failure. CONCLUSIONS: Increased enamel thickness, increased porcelain thickness, and increased combined enamel and porcelain thickness all profoundly raised the failure loads necessary to cause catastrophic failure. Enamel and feldspathic porcelain behaved in a like manner. Surface contact damage occurred initially. Final catastrophic failure followed flexural radial cracking. Bonded porcelain veneers were highly damage tolerant.

A scalable system for production of functional pancreatic progenitors from human embryonic stem cells.

Development of a human embryonic stem cell (hESC)-based therapy for type 1 diabetes will require the translation of proof-of-principle concepts into a scalable, controlled, and regulated cell manufacturing process. We have previously demonstrated that hESC can be directed to differentiate into pancreatic progenitors that mature into functional glucose-responsive, insulin-secreting cells in vivo. In this study we describe hESC expansion and banking methods and a suspension-based differentiation system, which together underpin an integrated scalable manufacturing process for producing pancreatic progenitors. This system has been optimized for the CyT49 cell line. Accordingly, qualified large-scale single-cell master and working cGMP cell banks of CyT49 have been generated to provide a virtually unlimited starting resource for manufacturing. Upon thaw from these banks, we expanded CyT49 for two weeks in an adherent culture format that achieves 50-100 fold expansion per week. Undifferentiated CyT49 were then aggregated into clusters in dynamic rotational suspension culture, followed by differentiation en masse for two weeks with a four-stage protocol. Numerous scaled differentiation runs generated reproducible and defined population compositions highly enriched for pancreatic cell lineages, as shown by examining mRNA expression at each stage of differentiation and flow cytometry of the final population. Islet-like tissue containing glucose-responsive, insulin-secreting cells was generated upon implantation into mice. By four- to five-months post-engraftment, mature neo-pancreatic tissue was sufficient to protect against streptozotocin (STZ)-induced hyperglycemia. In summary, we have developed a tractable manufacturing process for the generation of functional pancreatic progenitors from hESC on a scale amenable to clinical entry.

Resinous denture base fracture resistance: effects of thickness and teeth.

PURPOSE: Fracture is a frequent complication of resinous prostheses. The purpose of this study was to evaluate the effect of thickness on flexural strength of a resinous prosthesis containing a prosthetic tooth. MATERIALS AND METHODS: Beam-shaped specimens 65-mm long, 12-mm wide, and 1, 2, 3, 4, or 6 mm in thickness were made from high-impact strength polymethyl methacrylate denture base material, each containing a resin-based molar prosthetic tooth at the center of the beam. A group of 3-mm-thick specimens without a prosthetic tooth (n = 7) were also made. Specimens were aged artificially, loaded in three-point flexure, examined fractographically, and analyzed. RESULTS: The 1- and 2-mm-thick beams underwent considerable deformation at low loads. Maximum loads varied considerably from 0.6 kg (1-mm beams) to 38 kg (6-mm beams). The 3-, 4-, and 6-mm beam groups all underwent brittle fracture, with mean relative flexural strengths of approximately 73 MPa. Denture teeth reduced the relative flexural strength of resin beams by 0.7 x. Fracture initiation sites were generally at tiny surface defects, but did not directly involve denture teeth. Denture resin fracture toughness was 3.2 MPa m1/2, and modulus of rupture was 104 MPa. CONCLUSION: Denture teeth substantially decreased the strength of resinous beams. Increased thickness markedly increased the load-bearing capacity of resinous beams containing denture teeth. Beams less than 2 mm in thickness with denture teeth were weakened substantially more than comparable beams of 2 mm or more in thickness. Surface finish was of critical importance. Fracture toughness was calculated fractographically, facilitating future forensic examination of clinically failed resinous prostheses.

Concerns with AED conversion: comparison of patient and physician perspectives.

When discussing AED conversion in the clinic, both the patient and physician perspectives on the goals and risks of this change are important to consider. To identify patient-reported and clinician-perceived concerns, a panel of epilepsy specialists was questioned about the topics discussed with patients and the clinician's perspective of patient concerns. Findings of a literature review of articles that report patient-expressed concerns regarding their epilepsy and treatment were also reviewed. Results showed that the specialist panel appropriately identified patient-reported concerns of driving ability, medication cost, seizure control, and medication side effects. Additionally, patient-reported concerns of independence, employment issues, social stigma, medication dependence, and undesirable cognitive effects are important to address when considering and initiating AED conversion.

Chemoattractant signaling between tumor cells and macrophages regulates cancer cell migration, metastasis and neovascularization.

Tumor-associated macrophages are known to influence cancer progression by modulation of immune function, angiogenesis, and cell metastasis, however, little is known about the chemokine signaling networks that regulate this process. Utilizing CT26 colon cancer cells and RAW 264.7 macrophages as a model cellular system, we demonstrate that treatment of CT26 cells with RAW 264.7 conditioned medium induces cell migration, invasion and metastasis. Inflammatory gene microarray analysis indicated CT26-stimulated RAW 264.7 macrophages upregulate SDF-1alpha and VEGF, and that these cytokines contribute to CT26 migration in vitro. RAW 264.7 macrophages also showed a robust chemotactic response towards CT26-derived chemokines. In particular, microarray analysis and functional testing revealed CSF-1 as the major chemoattractant for RAW 264.7 macrophages. Interestingly, in the chick CAM model of cancer progression, RAW 264.7 macrophages localized specifically to the tumor periphery where they were found to increase CT26 tumor growth, microvascular density, vascular disruption, and lung metastasis, suggesting these cells home to actively invading areas of the tumor, but not the hypoxic core of the tumor mass. In support of these findings, hypoxic conditions down regulated CSF-1 production in several tumor cell lines and decreased RAW 264.7 macrophage migration in vitro. Together our findings suggest a model where normoxic tumor cells release CSF-1 to recruit macrophages to the tumor periphery where they secrete motility and angiogenic factors that facilitate tumor cell invasion and metastasis.

Leucocyte recruitment under fluid shear: mechanical and molecular regulation within the inflammatory synapse.

1. Nature has evolved an exquisite system for regulation of leucocyte recruitment at sites of tissue inflammation. Mechanical energy translated to the red and white blood cells transports them from large arteries down to the microcirculation. 2. Neutrophils overcome the drag forces of blood flow by forming selectin and integrin adhesive bonds with the endothelium that coats the vessel wall. Leucocyte adhesion receptors have evolved unique mechanical and chemical properties that optimize for sequential binding and uptake of traction forces. 3. In the present brief review, we address how dispersive forces acting on a neutrophil in shear flow function to stabilize and synchronize bond formation within a macromolecular membrane complex we denote the inflammatory synapse.

Direct economic effect following mupirocin therapy failure among patients with uncomplicated skin and skin-structure infections in a large population-based cohort.

The goal of the study was to derive initial costs associated with failure of initial mupirocin therapy among patients diagnosed with uncomplicated skin and skin-structure infections (uSSSIs). A retrospective observational analysis of medical, pharmacy, and enrollment records was conducted using data from the National Managed Care Benchmark Database. Patients were classified as failing treatment with mupirocin if they either filled a second antibiotic commonly used to treat uSSSIs five to 30 days after their index mupirocin prescription fill or experienced a uSSSI-related hospitalization within 30 days after the index mupirocin prescription fill. Among 12,650 failure episodes, 11,867 (93.8%) required a second antibiotic contributing a mean cost of $62 per prescription. Approximately 4,782 (37.8%) had an associated outpatient encounter resulting in a mean cost of $221 per encounter. Nine percent of failures required a hospitalization with a mean cost of $6,597 per hospitalization. These medical, hospital, and pharmacy costs translated into an expected cost of $735.45 per mupirocin failure among patients with uSSSIs. The management of uSSSIs is costly in terms of health care resource use and direct health care expenditures when initial therapy with mupirocin fails.

Impaired integrin-dependent function in Wiskott-Aldrich syndrome protein-deficient murine and human neutrophils.

Wiskott-Aldrich syndrome (WAS) is a primary immunodeficiency that manifests as increased susceptibility to many pathogens. Although the spectrum of infections suffered by WAS patients is consistent with defects in neutrophil (PMN) function, the consequences of WAS protein (WASp) deficiency on this innate immune cell have been unclear. We report that deficiency of WASp in both human and murine PMNs resulted in profound defects in clustering of beta2 integrins, leading to defective adhesion and transendothelial migration under conditions of physiologic shear flow. Wild-type PMNs redistributed clustered beta2 integrins to the uropod of the cell during active migration, whereas WASp-deficient cells remain unpolarized. The WASp-deficient PMNs also showed reduced integrin-dependent activation of degranulation and respiratory burst. PMNs from a WAS patient manifested similar defects in integrin clustering and signaling. These results suggest that impaired beta2 integrin function in WASp-deficient PMNs may contribute substantially to the clinical immunodeficiency suffered by WAS patients.

Dynamic shifts in LFA-1 affinity regulate neutrophil rolling, arrest, and transmigration on inflamed endothelium.

Polymorphonuclear leukocyte (PMN) recruitment to vascular endothelium during acute inflammation involves cooperation between selectins, G-proteins, and beta2-integrins. LFA-1 (CD11a/CD18) affinity correlates with specific adhesion functions because a shift from low to intermediate affinity supports rolling on ICAM-1, whereas high affinity is associated with shear-resistant leukocyte arrest. We imaged PMN adhesion on cytokine-inflamed endothelium in a parallel-plate flow chamber to define the dynamics of beta2-integrin function during recruitment and transmigration. After arrest on inflamed endothelium, high-affinity LFA-1 aligned along the uropod-pseudopod major axis, which was essential for efficient neutrophil polarization and subsequent transmigration. An allosteric small molecule inhibitor targeted to the I-domain stabilized LFA-1 in an intermediate-affinity conformation, which supported neutrophil rolling but inhibited cell polarization and abrogated transmigration. We conclude that a shift in LFA-1 from intermediate to high affinity during the transition from rolling to arrest provides the contact-mediated signaling and guidance necessary for PMN transmigration on inflamed endothelium.

Molecular mechanics and dynamics of leukocyte recruitment during inflammation.

Discovery of new genes and proteins directly supporting leukocyte adhesion is waning, whereas there is heightened interest in the cell mechanics and receptor dynamics that lead from transient tethering via selectins to affinity shifts and adhesion strengthening through integrins. New optical tools enable real-time imaging of leukocyte rolling and arrest in parallel plate flow channels (PPFCs), and detection of single-molecule force spectroscopy provides an inner view of the intercellular adhesive contact region. Leukocyte recruitment during acute inflammation is triggered by ligation of G protein-coupled chemotactic receptors (GPCRs) and clustering of selectins. This, in turn, activates beta(2)-integrin (CD18), which facilitates cell capture and arrest in shear flow. This review provides a conceptual model for the molecular events supporting leukocyte recruitment.

Rolling dynamics of a neutrophil with redistributed L-selectin.

The most common white blood cell is the neutrophil, which slowly rolls along the walls of blood vessels due to the coordinated formation and breakage of chemical selectin-carbohydrate bonds. We show that L-selectin receptors are rapidly redistributed to form a cap at one end of the cell membrane during rolling via selectins or chemotactic stimulation. This topography significantly alters the adhesive dynamics as demonstrated by computer simulations of neutrophils rolling on a carbohydrate selectin-ligand substrate under flow. It was found that neutrophils with a redistributed L-selectin cap roll on sialyl Lewis-x with a quasi-periodic motion, as characterized by relatively low velocity intervals interspersed with regular jumps in the rolling velocity. On average, neutrophils with redistributed L-selectin rolled at a lower velocity when compared with cells having a uniform L-selectin distribution of equal average density. We speculate on the possible biological implications that these differences in adhesion dynamics will have during the inflammatory response.

Cytoskeletal interactions regulate inducible L-selectin clustering.

L-selectin (CD62L) amplifies neutrophil capture within the microvasculature at sites of inflammation. Activation by G protein-coupled stimuli or through ligation of L-selectin promotes clustering of L-selectin and serves to increase its adhesiveness, signaling, and colocalization with beta(2)-integrins. Currently, little is known about the molecular process regulating the lateral mobility of L-selectin. On neutrophil stimulation, a progressive change takes place in the organization of its plasma membrane, resulting in membrane domains that are characteristically enriched in glycosyl phosphatidylinositol (GPI)-anchored proteins and exclude the transmembrane protein CD45. Clustering of L-selectin, facilitated by E-selectin engagement or antibody cross-linking, resulted in its colocalization with GPI-anchored CD55, but not with CD45 or CD11c. Disrupting microfilaments in neutrophils or removing a conserved cationic motif in the cytoplasmic domain of L-selectin increased its mobility and membrane domain localization in the plasma membrane. In addition, the conserved element was critical for L-selectin-dependent tethering under shear flow. Our data indicate that L-selectin's lateral mobility is regulated by interactions with the actin cytoskeleton that in turn fortifies leukocyte tethering. We hypothesize that both membrane mobility and stabilization augment L-selectin's effector functions and are regulated by dynamic associations with membrane domains and the actin cytoskeleton.

Shear-dependent capping of L-selectin and P-selectin glycoprotein ligand 1 by E-selectin signals activation of high-avidity beta2-integrin on neutrophils.

Two adhesive events critical to efficient recruitment of neutrophils at vascular sites of inflammation are up-regulation of endothelial selectins that bind sialyl Lewis(x) ligands and activation of beta(2)-integrins that support neutrophil arrest by binding ICAM-1. We have previously reported that neutrophils rolling on E-selectin are sufficient for signaling cell arrest through beta(2)-integrin binding of ICAM-1 in a process dependent upon ligation of L-selectin and P-selectin glycoprotein ligand 1 (PSGL-1). Unresolved are the spatial and temporal events that occur as E-selectin binds to human neutrophils and dynamically signals the transition from neutrophil rolling to arrest. Here we show that binding of E-selectin to sialyl Lewis(x) on L-selectin and PSGL-1 drives their colocalization into membrane caps at the trailing edge of neutrophils rolling on HUVECs and on an L-cell monolayer coexpressing E-selectin and ICAM-1. Likewise, binding of recombinant E-selectin to PMNs in suspension also elicited coclustering of L-selectin and PSGL-1 that was signaled via mitogen-activated protein kinase. Binding of recombinant E-selectin signaled activation of beta(2)-integrin to high-avidity clusters and elicited efficient neutrophil capture of beta(2)-integrin ligands in shear flow. Inhibition of p38 and p42/44 mitogen-activated protein kinase blocked the cocapping of L-selectin and PSGL-1 and the subsequent clustering of high-affinity beta(2)-integrin. Taken together, the data suggest that E-selectin is unique among selectins in its capacity for clustering sialylated ligands and transducing signals leading to neutrophil arrest in shear flow.

Topographic requirements and dynamics of signaling via L-selectin on neutrophils.

Cross-linking of L-selectin on leukocytes signals phosphorylation of mitogen-activated protein kinases (MAPKs) leading to activation of CD18 function and enhanced transmigration on inflamed endothelium. We examined how alterations in the topography of L-selectin correlate with the dynamics of CD18 activation and phosphorylation of MAPK. Simultaneous ligation of humanized antibodies DREG55 and DREG200 provided a strategy for regulating the extent of cross-linking. Triggering of CD11b/CD18 upregulation and adhesion required clustering of L-selectin to microvillus-sized patches of approximately 0.2 microm(2). Immunofluorescence revealed that L-selectin was colocalized with high-affinity CD18. Anti-L-selectin-coated protein A microspheres indicated that a single site of contact to a 5.5-microm bead, or multiple contacts to 0.94- or 0.3-microm beads, elicited maximum neutrophil activation. Adhesion signaled via L-selectin coincided with the kinetics of MAPK phosphorylation and was inhibited by blocking p38 or p42/44 activity. These data demonstrate the capacity of L-selectin to transduce signals effecting rapid ( approximately 1 s) neutrophil adhesion that is regulated by the size and frequency of receptor clustering.

Dynamic regulation of LFA-1 activation and neutrophil arrest on intercellular adhesion molecule 1 (ICAM-1) in shear flow.

Neutrophil recruitment during acute inflammation is triggered by G-protein-linked chemotactic receptors that in turn activate beta(2) integrin (CD18), deemed a critical step in facilitating cell capture and arrest under the shear force of blood flow. A conformational switch in the I domain allosteric site (IDAS) and in CD18 regulates LFA-1 affinity for endothelial ligands including intercellular adhesion molecule 1 (ICAM-1). We examined the dynamics of CD18 activation in terms of the efficiency of neutrophil capture of ICAM-1, and we correlated this with the membrane topography of 327C, an antibody that recognizes the active conformation of CD18 I-like domain. Adhesion increased in direct proportion to chemotactic stimulus rising 7-fold over a log range of interleukin-8 (IL-8). A threshold dose of approximately 75 pm IL-8, corresponding to ligation of only approximately 10-100 receptors, was sufficient to activate approximately 20,000 CD18 and a rapid boost in the capture efficiency on ICAM-1. This was accompanied by a rapid redistribution of active LFA-1, but not Mac-1, into membrane patches, a necessary component for optimum adhesion efficiency. Shear-resistant arrest on a monolayer of ICAM-1 was reversed within minutes of chemotactic stimulation correlating with a shift from high to low affinity CD18 and dispersal of patches of active CD18. Mobility of active CD18 into high avidity patches was dependent on phosphatidylinositol 3-kinase activity and not F-actin polymerization. The data reveal that the number of chemotactic receptors bound and the topography and lifetime of high affinity LFA-1 tightly regulate the efficiency of neutrophil capture on ICAM-1.