Rate-dependent changes in action potential duration and membrane currents in hamster ventricular myocytes

Hamsters are frequently studied as a model of cardiomyopathy, but the electrophysiological properties of a hamster heart are not well defined. We examined rate-dependent changes in action potentials and underlying ionic mechanisms in isolated ventricular myocytes from hamster hearts using the whole-cell configuration of the patch clamp technique. At 0.1 Hz stimulation, the mean action potential duration at 90% (APD90) and 20% (APD20) repolarization were 63+/-7 ms and 9+/-1 ms, respectively ( n=17). With increasing frequency of stimulation, APD progressively prolonged to 119+/-16 ms (APD90) and 36+/-7 ms (APD20) at 6.0 Hz. A further increase in the rate of stimulation to 8.0 Hz did not change APD significantly. Application of 4 mM 4-aminopyridine (4-AP) lengthened APD markedly and completely prevented the rate-dependent prolongation. Cd2+ (0.2 mM) shortened APD and generally attenuated the rate-dependent lengthening of APD up to 5.0 Hz, but unaffected the lengthening of APD with the further increase in the rate. At plateau voltages, there were two time-dependent currents, Ito1 and I(Ca,L). Recovery from inactivation for Ito1 had two components: t(slow)=980+/-129 ms accounting for 58% of the total fraction, and t(fast)=39+/-13 ms ( n=7). Recovery from inactivation for I(Ca,L) was rapid with t=20+/-4 ms ( n=6). Results suggest that the slow recovery from inactivation in Ito1 is the main reason for the rate-dependent prolongation of APD in hamster ventricular myocytes.     

Enzymatic digestion of adult human articular cartilage yields a small fraction of the total available cells

We investigated whether different protocols for the digestion of adult human articular cartilage influence the cell yield and capacity to attach and proliferate in culture dishes. Chondrocyte yields were expressed as a percentage of the total number of cells in the tissue, determined both histologically (using the dissector method) and biochemically (measuring the DNA content of tissue digests). Human cartilage specimens (n = 79) were digested using different protocols based on combinations of collagenase II (CGN), trypsin/EDTA, hyaluronidase, and tosyllysylchloromethane (TLCM). Yields of viable chondrocytes were the highest within a specific range of CGN concentrations and digestion times, but always < 22% of the total available cells. The combination of CGN with trypsin/EDTA or TLCM accelerated the digestion process but did not significantly increase cell yields. The percentage of viable cells that attached to culture dishes ranged 75-85% (< 19% of the total) and was reduced by TLCM. Doubling times of attached cells were comparable in all experimental groups. Our results indicate that chondrocyte yields and capacity to attach and proliferate are not highly sensitive to the specific isolation protocol used. However, typically used cartilage digestion protocols yield only a small fraction of the total available cells, possibly introducing an uncontrolled selection of certain chondrocyte subpopulations.     

Custom step wedge blocking using dynamic multileaf collimation for parametrial pelvic boost irradiation following brachytherapy for carcinoma of the cervix

Carcinoma of the cervix is typically treated with a combination of intracavitary brachytherapy and external beam radiation. The external beam dose is delivered with whole pelvis fields followed by split fields that protect midline organs at risk (bladder and rectum) while treating the parametria. Three approaches have been developed to shield midline structures: a simple rectangular block, a block customized to a single brachytherapy isodose line, and a step wedge filter constructed to conform to multiple brachytherapy isodose lines. A customized step wedge filter has the potential to produce a more homogeneous dose distribution but has not achieved widespread use due to labor intensive construction. We have developed a simple, novel method to produce a custom midline step wedge using dynamic multileaf collimation (dMLC). A comparison of film measurements in a phantom with the dose calculated by a commercial treatment planning system demonstrated agreement within 3% or 3 mm. The technique requires delivery times comparable to conventional techniques.     

Validation of target volume and position in respiratory gated CT planning and treatment

The capability of a commercial respiratory gating system based on video tracking of reflective markers to reduce motion-induced CT planning and treatment errors was evaluated. Spherical plastic shells (2.8-82 cm3), simulating the gross target volume (GTV), were placed in a water-filled body phantom that was moved sinusoidally along the longitudinal axis of the CT scanner and the accelerator for +/- 1 cm at 15-30 cycle/min. During gated CT imaging, the x-ray exposure was initiated by the gating system shortly before the end of expiration (so that the imaging time would be centered at the end of expiration); it was terminated by the scanner after completion of each slice. In nongated CT images, the target appeared distorted and often broken up. GTVs volume errors ranged 16%-110% in axial scans, and 7%-36% in spiral scans. In gated CT images, the spheres appeared 3 and 5 mm longer than their actual diameters (volume errors 2%-16%), at the respective respiration rates of 15 and 20 cycles/min. At 30 cycles/min the target appeared 1 cm longer, and volume error ranged 25%-53%. During treatment, gating kept the beam on for a duration equal to the CT acquisition time of 1 s/slice. The difference in positional errors between gated CT and portal films was 1 mm, regardless the size of residual motion errors. Because of the potential of suboptimal placement of the gating window between CT imaging and treatment, an extra 1.5-2.5 mm safety margin can be added regardless of the size of residual motion error. For respiratory rates > or = 30 cycles/min, the effectiveness of gating is limited by large residual motion in the 1 s CT acquisition time.     

Triglycidylamine crosslinking of porcine aortic valve cusps or bovine pericardium results in improved biocompatibility, biomechanics, and calcification resistance: chemical and biological mechanisms

We investigated a novel polyepoxide crosslinker that was hypothesized to confer both material stabilization and calcification resistance when used to prepare bioprosthetic heart valves. Triglycidylamine (TGA) was synthesized via reacting epichlorhydrin and NH(3). TGA was used to crosslink porcine aortic cusps, bovine pericardium, and type I collagen. Control materials were crosslinked with glutaraldehyde (Glut). TGA-pretreated materials had shrink temperatures comparable to Glut fixation. However, TGA crosslinking conferred significantly greater collagenase resistance than Glut pretreatment, and significantly improved biomechanical compliance. Sheep aortic valve interstitial cells grown on TGA-pretreated collagen did not calcify, whereas sheep aortic valve interstitial cells grown on control substrates calcified extensively. Rat subdermal implants (porcine aortic cusps/bovine pericardium) pretreated with TGA demonstrated significantly less calcification than Glut pretreated implants. Investigations of extracellular matrix proteins associated with calcification, matrix metalloproteinases (MMPs) 2 and 9, tenascin-C, and osteopontin, revealed that MMP-9 and tenascin-C demonstrated reduced expression both in vitro and in vivo with TGA crosslinking compared to controls, whereas osteopontin and MMP-2 expression were not affected. TGA pretreatment of heterograft biomaterials results in improved stability compared to Glut, confers biomechanical properties superior to Glut crosslinking, and demonstrates significant calcification resistance.     

Investigation of the morphology of cell clones, derived from the mammalian EBTr cell line and their susceptibility to vaccine avian poxvirus strains FK and Dessau

The ability for replication of vaccine avian pox viral strains FK and Dessau in cell clones, derived from the EBTr cell line, derived from embryonic bovine trachea, was studied. The derived seven cell clones showed different morphological characteristics and diverse sensitivity to both vaccine avian pox viral strains. Hence, the EBTr-derived cell clones could be used for cultivation, as well as for differentiation of vaccine avian pox viral strains. In addition, studies have been undertaken to elucidate the possible use of cultivated strains in these heterologous cell culture system's vaccine avian pox viral strains for biotechnology, as well as for solving problems, related to infection of people with avian viruses.     

Activated polyurethane modified with latent thiol groups

A novel type of modified polyurethane with pendant acetylthio groups (as a latent form of thiol groups) has been proposed for the use in surface modifications with various biomolecules. The polymer was prepared via a modified variant of low-temperature bromoalkylation of urethane hard segments followed by the reaction of pendant bromoalkyl groups with thiolacetic acid in mild conditions. The extent of modification with acetylthio groups can be made as high as 0.45 mmol/g. After deprotection of acetylthio groups and reaction of the resulting thiol groups with an excess of Ellman's reagent, 0.1 nmol/cm(2) of thiol-reactive 3-carboxy-4-nitrophenyldithio groups were detected on the surface of films cast from the modified polymer. A sensitive fluorescent probe--dansyl-L-cysteine was used for the quantification of thiol-reactive groups bound to the surface. The acetylthio-modified polyurethane is sufficiently stable to withstand conditions typical for the high-temperature processing (molding, extrusion) of polyurethanes.