Proof of concept testing of a positive reference material for in vivo and in vitro sensitization testing of medical devices.

In vivo skin sensitization tests are required to evaluate the biological safety of medical devices in contact with living organisms to provide safe medical care to patients. Negative and positive reference materials have been developed for biological tests of cytotoxicity, implantation, hemolysis, and in vitro skin irritation. However, skin sensitization tests are lacking. In this study, polyurethane sheets containing 1 wt/wt % 2,4-dinitrochlorobenzene (DNCB-PU) were developed and evaluated as a positive reference material for skin sensitization tests. DNCB-PU sheet extracts prepared with sesame oil elicited positive sensitization responses for in vivo sensitization potential in the guinea pig maximization test and the local lymph node assay. Furthermore, DNCB-PU sheet extracts prepared with water and acetonitrile, 10% fetal bovine serum-containing medium, or sesame oil elicited positive sensitization responses as alternatives to animal testing based on the amino acid derivative reactivity assay, human cell line activation test, and epidermal sensitization assay, respectively. These data suggest that the DNCB-PU sheet is an effective extractable positive reference material for in vivo and in vitro skin sensitization testing in medical devices. The formulation of this reference material will lead to the development of safer medical devices that contribute to patient safety.

Poly(2-methoxyethyl acrylate) (PMEA) improves the thromboresistance of FRED flow diverters: a thrombogenic evaluation of flow diverters with human blood under flow conditions.

BACKGROUND: Surface modification of flow-diverting stents has been explored to reduce thrombus-related complications that may arise under clinical use. This study investigated the thromboresistant properties of the flow redirection endoluminal device (FRED) X, a flow diverter treated with a copolymer of poly(2-methoxyethyl acrylate) (PMEA; X Technology). METHODS: The performance of FRED, FRED X, and Pipeline Flex with Shield Technology (sPED) was evaluated in an in vitro blood loop model. Blood activation level was assessed by the concentration of thrombin-antithrombin complex (TAT), ß-thromboglobulin (ß-TG), and platelet count, and qualitatively by scanning electron microscopy (SEM). Cellular adhesion characteristics were measured using human aortic endothelial cells that were seeded on flat sheets mimicking the surface of FRED, FRED X, and sPED, and evaluated with fluorescence microscopy. Statistical comparisons were conducted using one-way analysis of variance (ANOVA) with Tukey post hoc tests. RESULTS: FRED X, sPED, and control blood loops showed significantly reduced blood activation levels (TAT and ß-TG) compared with FRED (p<0.01). Consequently, FRED showed a significant decrease in platelet count compared with FRED X, sPED, and control loops (p<0.01). SEM imaging showed the lowest accumulation of blood cell-like deposits on FRED X compared with sPED and FRED, while FRED had the highest accumulation. Endothelial cells adhered and were widely spread on X Technology-treated sheets, while minimal cell adhesion was observed on phosphorylcholine-treated sheets. CONCLUSION: The X Technology surface modification of FRED X demonstrated superior thromboresistant properties over untreated FRED while maintaining comparable cellular adhesion. Taken together, these properties may help mitigate material-related thromboembolic complications.

Structural analysis and identification of novel isoforms of the circadian clock gene period in the silk moth Bombyx mori.

The molecular basis of the circadian clock is an autoregulatory feedback loop in which the PAS domain-containing protein PERIOD periodically inhibits its own transcription. In the present study on PERIOD of the silk moth Bombyx mori, we have cloned two distinct period mRNA homologues with different PAS domain sequences either with or without the pentapeptide GTQEK. A period cDNA fragment first amplified by PCR exhibited a 15 bp-deleted nucleotide sequence in the PAS domain, compared with the database sequence. A possible alternative splicing mechanism was examined by PCR analyses, and a 15 bp-inserted clone was also amplified. The entire sequences of these period alpha and period beta isoforms were then determined by the 3' and 5' RACE methods. Isoform period alpha consists of a 3,324 bp oligonucleotide encoding 1,108 amino acid residues, whereas isoform period beta comprises 3,309 bp corresponding to 1,103 amino acids. Isoforms period alpha and period beta were found to be exactly identical except for the 15 bp deletion/insertion site. Such a pair of isoforms with a deletion/insertion sequence, namely two splice variants, has previously been reported only for the PERIOD proteins of the two honeybees, Apis mellifera and A. cerana. The occurrence of an alternative splicing mechanism in the B. mori period gene was hypothesized based on the genome structure recently clarified. Bombyx mori PERIOD alpha and beta proteins are the isomers that reveal firstly the different PAS domain sequences.