There is a strong relationship between mechanical stress and calcification in biological prosthetic heart valves. A dynamic in vitro calcification test has been used to study the relationship between stress distributions in the leaflets of bovine pericardial valves and the deposition of calcium over the leaflet surfaces. Intuitive stress regions have been defined over the leaflet surfaces. Calcium uptake by the leaflets has been assayed directly by ashing of leaflet material and analysis of the ash by atomic absorption spectrophotometry. Calcium and phosphorus distribution over the leaflet surface has been analyzed using energy-dispersive x-ray analysis by scanning electron microscope and data points assigned to the appropriate stress region. The uptake of calcium is assessed by comparing stress regions, surfaces, and the degree of calcification of the valve. Differences between stress regions and surfaces are significant. Uptake of calcium in these valves appears to be strongly related to the degree and type of stress present in the valve leaflets.
This study has examined a range of methods of studying the calcification process in bovine pericardial and polyurethane biomaterials. The calcification methods include static and dynamic, in vitro and in vivo tests. The analytical methods include measurement of depletion rates of calcium and phosphate from in vitro calcifying solutions, analysis of tissue contents of calcium, histological staining of tissue sections for calcium, X-ray elemental analysis, by scanning electron microscopy, of calcium and phosphorus distributions over valve leaflets calcified in vitro under dynamic conditions. Bovine pericardium, in all test settings, calcified to a much greater degree than polyurethane biomaterials. Polyurethane extracts calcified to a greater degree than bulk polyurethanes. The test protocol used allows progress through increasingly demanding calcification tests, with the possibility of eliminating unsuitable materials with tests of limited complexity and expense.
1Synthesis of homogenous glycans in quantitative yields represents a major bottleneck to the 2 production of molecular tools for glycoscience, such as glycan microarrays, affinity resins, and 3 reference standards. Here, we describe a combined biological/enzymatic method termed 4 bioenzymatic synthesis that is capable of efficiently converting microbially-derived precursor 5 oligosaccharides into structurally uniform human-type N-glycans. Unlike starting material 6 obtained by chemical synthesis or direct isolation from natural sources, which can be time 7consuming and costly to generate, bioenzymatic synthesis involves precursors derived from 8 renewable sources including wild-type Saccharomyces cerevisiae glycoproteins and lipid-linked 9 oligosaccharides from glycoengineered Escherichia coli. Following deglycosylation of these 10 biosynthetic precursors, the resulting microbial oligosaccharides are subjected to a greatly 11 simplified purification scheme followed by structural remodeling using commercially available 12 and recombinantly produced glycosyltransferases including key N-13 acetylglucosaminyltransferases (e.g., GnTI, GnTII, and GnTIV) involved in early remodeling of 14 glycans in the mammalian glycosylation pathway. Using this approach, preparative quantities of 15 hybrid and complex-type N-glycans including asymmetric multi-antennary structures were 16 generated all without the need of a specialized skillset. Collectively, our results reveal 17 bioenzymatic synthesis to be a user-friendly methodology for rapidly supplying homogeneous 18 oligosaccharide structures that can be used to understand the human glycome and probe the 19 biological roles of glycans in health and disease.
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