Ryan M. Jamiolkowski

A comprehensive review of topical hemostatic agents: efficacy and recommendations for use.

Since ancient times we have attempted to facilitate hemostasis by application of topical agents. In the last decade, the number of different effective hemostatic agents has increased drastically. In order for the modern surgeon to successfully choose the right agent at the right time, it is essential to understand the mechanism of action, efficacy and possible adverse events as they relate to each agent. In this article we provide a comprehensive review of the most commonly used hemostatic agents, subcategorized as physical agents, absorbable agents, biologic agents, and synthetic agents. We also evaluate novel hemostatic dressings and their application in the current era. Furthermore, wholesale acquisition prices for hospitals in the United States are provided to aid in cost analysis. We conclude with an expert opinion on which agent to use under different scenarios.

Anion Binding of Short, Flexible Aryl Triazole Oligomers

The flexible, electropositive cavity of linear 1,4-diaryl-1,2,3-triazole oligomers provides a suitable host for complexation of various anions. The binding affinities for various combinations of oligomer and anion were determined by (1)H NMR titrations. Effective ionic radius is found to be a primary determinant of the relative binding interactions of various guests, with small but measurable deviations in the case of nonspherical anions. Solvent effects are significant, and the strength of the binding interaction is found to depend directly on the donor ability of the solvent. A picture emerges in which anion binding can be effectively interpreted in terms of a competition between two solvation spheres: one provided by the solvent and a second dominated by a folded cavity lined with electropositive 1,2,3-triazole CH protons. Implications for rigid macrocycles and other multivalent hosts are discussed.

The Biocompatibility of Titanium Cardiovascular Devices Seeded With Autologous Blood-Derived Endothelial Progenitor Cells: EPC-Seeded Antithrombotic Ti Implants

Implantable and extracorporeal cardiovascular devices are commonly made from titanium (Ti) (e.g. Ti-coated Nitinol stents and mechanical circulatory assist devices). Endothelializing the blood-contacting Ti surfaces of these devices would provide them with an antithrombogenic coating that mimics the native lining of blood vessels and the heart. We evaluated the viability and adherence of peripheral blood-derived porcine endothelial progenitor cells (EPCs), seeded onto thin Ti layers on glass slides under static conditions and after exposure to fluid shear stresses. EPCs attached and grew to confluence on Ti in serum-free medium, without preadsorption of proteins. After attachment to Ti for 15 min, less than 5% of the cells detached at a shear stress of 100 dyne / cm(2). Confluent monolayers of EPCs on smooth Ti surfaces (Rq of 10 nm), exposed to 15 or 100 dyne/cm(2) for 48 h, aligned and elongated in the direction of flow and produced nitric oxide dependent on the level of shear stress. EPC-coated Ti surfaces had dramatically reduced platelet adhesion when compared to uncoated Ti surfaces. These results indicate that peripheral blood-derived EPCs adhere and function normally on Ti surfaces. Therefore EPCs may be used to seed cardiovascular devices prior to implantation to ameliorate platelet activation and thrombus formation.

Use of autologous blood-derived endothelial progenitor cells at point-of-care to protect against implant thrombosis in a large animal model.

Titanium (Ti) is commonly utilized in many cardiovascular devices, e.g. as a component of Nitinol stents, intra- and extracorporeal mechanical circulatory assist devices, but is associated with the risk of thromboemboli formation. We propose to solve this problem by lining the Ti blood-contacting surfaces with autologous peripheral blood-derived late outgrowth endothelial progenitor cells (EPCs) after having previously demonstrated that these EPCs adhere to and grow on Ti under physiological shear stresses and functionally adapt to their environment under flow conditions ex vivo. Autologous fluorescently-labeled porcine EPCs were seeded at the point-of-care in the operating room onto Ti tubes for 30 min and implanted into the pro-thrombotic environment of the inferior vena cava of swine (n = 8). After 3 days, Ti tubes were explanted, disassembled, and the blood-contacting surface was imaged. A blinded analysis found all 4 cell-seeded implants to be free of clot, whereas 4 controls without EPCs were either entirely occluded or partially thrombosed. Pre-labeled EPCs had spread and were present on all 4 cell-seeded implants while no endothelial cells were observed on control implants. These results suggest that late outgrowth autologous EPCs represent a promising source of lining Ti implants to reduce thrombosis in vivo.

Parallel-plate Flow Chamber and Continuous Flow Circuit to Evaluate Endothelial Progenitor Cells under Laminar Flow Shear Stress

The overall goal of this method is to describe a technique to subject adherent cells to laminar flow conditions and evaluate their response to well quantifiable fluid shear stresses. Our flow chamber design and flow circuit (Fig. 1) contains a transparent viewing region that enables testing of cell adhesion and imaging of cell morphology immediately before flow (Fig. 11A, B), at various time points during flow (Fig. 11C), and after flow (Fig. 11D). These experiments are illustrated with human umbilical cord blood-derived endothelial progenitor cells (EPCs) and porcine EPCs. This method is also applicable to other adherent cell types, e.g. smooth muscle cells (SMCs) or fibroblasts. The chamber and all parts of the circuit are easily sterilized with steam autoclaving. In contrast to other chambers, e.g. microfluidic chambers, large numbers of cells (> 1 million depending on cell size) can be recovered after the flow experiment under sterile conditions for cell culture or other experiments, e.g. DNA or RNA extraction, or immunohistochemistry (Fig. 11E), or scanning electron microscopy. The shear stress can be adjusted by varying the flow rate of the perfusate, the fluid viscosity, or the channel height and width. The latter can reduce fluid volume or cell needs while ensuring that one-dimensional flow is maintained. It is not necessary to measure chamber height between experiments, since the chamber height does not depend on the use of gaskets, which greatly increases the ease of multiple experiments. Furthermore, the circuit design easily enables the collection of perfusate samples for analysis and/or quantification of metabolites secreted by cells under fluid shear stress exposure, e.g. nitric oxide (Fig. 12).

Increased yield of endothelial cells from peripheral blood for cell therapies and tissue engineering.

AIM Peripheral blood-derived endothelial cells (pBD-ECs) are an attractive tool for cell therapies and tissue engineering, but have been limited by their low isolation yield. We increase pBD-EC yield via administration of the chemokine receptor type 4 antagonist AMD3100, as well as via a diluted whole blood incubation (DWBI). MATERIALS & METHODS Porcine pBD-ECs were isolated using AMD3100 and DWBI and tested for EC markers, acetylated LDL uptake, growth kinetics, metabolic activity, flow-mediated nitric oxide production and seeded onto titanium tubes implanted into vessels of pigs. RESULTS DWBI increased the yield of porcine pBD-ECs 6.6-fold, and AMD3100 increased the yield 4.5-fold. AMD3100-mobilized ECs were phenotypically indistinguishable from nonmobilized ECs. In porcine implants, the cells expressed endothelial nitric oxide synthase, reduced thrombin-antithrombin complex systemically and prevented thrombosis. CONCLUSION Administration of AMD3100 and the DWBI method both increase pBD-EC yield.

Regenerating titanium ventricular assist device surfaces after gold/palladium coating for scanning electron microscopy

Titanium is one of the most commonly used materials for implantable devices in humans. Scanning electron microscopy (SEM) serves as an important tool for imaging titanium surfaces and analyzing cells and other organic matter adhering to titanium implants. However, high‐vacuum SEM imaging of a nonconductive sample requires a conductive coating on the surface. A gold/palladium coating is commonly used and to date no method has been described to “clean” such gold/palladium covered surfaces for repeated experiments without etching the titanium itself. This constitutes a major problem with titanium‐based implantable devices which are very expensive and thus in short supply. Our objective was to devise a protocol to regenerate titaniumsurfaces after SEM analysis. In a series of experiments, titanium samples from implantable cardiac assist devices were coated with fibronectin, seeded with cells and then coated with gold/palladium for SEM analysis. X‐ray photoelectron spectroscopy spectra were obtained before and after five different cleaning protocols. Treatment with aqua regia (a 1:3 solution of concentrated nitric and hydrochloric acid), with or without ozonolysis, followed by sonication in soap solution and sonication in deionized water, allowed regenerating titanium surfaces to their original state. Atomic force microscopy confirmed that the established protocol did not alter the titanium microstructure. The protocol described herein is applicable to almost all titanium surfaces used in biomedical sciences and because of its short exposure time to aqua regia, will likely work for many titanium alloys as well. Microsc. Res. Tech., 2009.

tRNA Fluctuations Observed on Stalled Ribosomes Are Suppressed during Ongoing Protein Synthesis

The pretranslocation complex of the ribosome can undergo spontaneous fluctuations of messenger RNA and transfer RNAs (tRNAs) between classical and hybrid states, and occupation of the hybrid tRNA positions has been proposed to precede translocation. The classical and hybrid state tRNA positions have been extensively characterized when the ribosome is stalled along the messenger RNA by either the absence or delayed addition of elongation factor G (EF-G), or by the presence of antibiotics or GTP analogs that block translocation. However, during multiple ongoing elongation cycles when both EF-G and ternary complexes are present, EF-G can bind to the pretranslocation complex much faster than the timescale of the classic-hybrid transitions. Using single-molecule fluorescence resonance energy transfer between adjacent tRNAs and between A-site tRNA and ribosomal protein L11, we found that the tRNAs do not fluctuate between the hybrid and classical states, but instead adopt a position with fluorescence resonance energy transfer efficiencies between those of the stalled classical and hybrid states.

Point-of-Care Rapid-Seeding Ventricular Assist Device with Blood-Derived Endothelial Cells to Create a Living Antithrombotic Coating.

The most promising alternatives to heart transplantation are left ventricular assist devices and artificial hearts; however, their use has been limited by thrombotic complications. To reduce these, sintered titanium (Ti) surfaces were developed, but thrombosis still occurs in approximately 7.5% of patients. We have invented a rapid-seeding technology to minimize the risk of thrombosis by rapid endothelialization of sintered Ti with human cord blood-derived endothelial cells (hCB-ECs). Human cord blood-derived endothelial cells were seeded within minutes onto sintered Ti and exposed to thrombosis-prone low fluid flow shear stresses. The hCB-ECs adhered and formed a confluent endothelial monolayer on sintered Ti. The exposure of sintered Ti to 4.4 dynes/cm2 for 20 hr immediately after rapid seeding resulted in approximately 70% cell adherence. The cell adherence was not significantly increased by additional ex vivo static culture of rapid-seeded sintered Ti before flow exposure. In addition, adherent hCB-ECs remained functional on sintered Ti, as indicated by flow-induced increase in nitric oxide secretion and reduction in platelet adhesion. After 15 day ex vivo static culture, the adherent hCB-ECs remained metabolically active, expressed endothelial cell functional marker thrombomodulin, and reduced platelet adhesion. In conclusion, our results demonstrate the feasibility of rapid-seeding sintered Ti with blood-derived hCB-ECs to generate a living antithrombotic surface.

A new in vitro assay measuring direct interaction of nonsense suppressors with the eukaryotic protein synthesis machinery

Nonsense suppressors (NonSups) treat premature termination codon (PTC) disorders by inducing the selection of near cognate tRNAs at the PTC position, allowing readthrough of the PTC and production of full-length protein. Studies of NonSup-induced readthrough of eukaryotic PTCs have been carried out using animals, cells or crude cell extracts. In these studies, NonSups can promote readthrough directly, by binding to components of the protein synthesis machinery, or indirectly, by inhibiting nonsense-mediated mRNA decay or by other mechanisms. Here we utilize a highly-purified in vitro system (Zhang et al., 2016. eLife 5: e13429) to measure exclusively direct NonSup-induced readthrough. Of 17 previously identified NonSups, 13 display direct effects, apparently via at least two different mechanisms. We can monitor such direct effects by single molecule FRET (smFRET). Future smFRET experiments will permit elucidation of the mechanisms by which NonSups stimulate direct readthrough, aiding ongoing efforts to improve the clinical usefulness of NonSups.