Single molecule investigation of the onset and minimum size of the calcium-mediated junction zone in alginate

One of the principal roles of alginate, both natively and in commercial applications, is gelation via Ca2+-mediated crosslinks between blocks of guluronic acid. In this work, single molecule measurements were carried out between well-characterised series of nearly monodisperse guluronic acid blocks (‘oligoGs’) using dynamic force spectroscopy. The measurements provide evidence that for interaction times on the order of tens of milliseconds the maximum crosslink strength is achieved by pairs of oligoGs long enough to allow the coordination of 4 Ca2+ ions, with both shorter and longer oligomers forming weaker links. Extending the interaction time from tens to hundreds of milliseconds allows longer oligoGs to achieve much stronger crosslinks but does not change the strength of individual links between shorter oligoGs. These results are considered in light of extant models for the onset of cooperative crosslinking in polyelectrolytes and an anisotropic distribution of oligoGs on interacting surfaces and provide a timescale for the formation and relaxation of alginate gels at the single crosslink level

New Hypothesis on the Role of Alternating Sequences in Calcium-Alginate Gels

The availability of mannuronan and mannuronan C-5 epimerases allows the production of a strictly alternating mannuronate-guluronate (MG) polymer and the MG-enrichment of natural alginates, providing a powerful tool for the analysis of the role of such sequences in the calcium-alginate gel network. In view of the calcium binding properties of long alternating sequences revealed by circular dichroism studies which leads eventually to the formation of stable hydrogels, their direct involvement in the gel network is here suggested. In particular, 1H NMR results obtained from a mixed alginate sample containing three polymeric species, G blocks, M blocks, and MG blocks, without chemical linkages between the block structures, indicate for the first time the formation of mixed junctions between G and MG blocks. This is supported by the analysis of the Young's modulus of hydrogels from natural and epimerized samples obtained at low calcium concentrations. Furthermore, the "zipping" of long alternating sequences in secondary MG/MG junctions is suggested to account for the shrinking (syneresis) of alginate gels in view of its dependence on the length of the MG blocks. As a consequence, a partial network collapse, macroscopically revealed by a decrease in the Young's modulus, occurred as the calcium concentration in the gel was increased. The effect of such "secondary" junctions on the viscoelastic properties of alginate gels was evaluated measuring their creep compliance under uniaxial compression. The experimental curves, fitted by a model composed of a Maxwell and a Voigt element in series, revealed an increase in the frictional forces between network chains with increasing length of the alternating sequences. This suggests the presence of an ion mediated mechanism preventing the shear of the gel

Assessment of insulin secretion in vitro from microencapsulated fetal porcine islet-like cell clusters and rat, mouse, and human pancreatic islets.

BACKGROUND: The possibility of transplanting microencapsulated pancreatic islets into patients with insulin-dependent diabetes mellitus, either as allografts or xenografts, has attracted great interest. A critical evaluation of the results obtained reveals that the success has been very limited. The aim of the present study was to compare the in vitro function of microencapsulated islets obtained from adult humans, adult mice, adult rats, and fetal pigs. METHODS: Human pancreatic islets were isolated at beta-Cell Transplant in Brussels, Belgium, and sent to the Department of Medical Cell Biology, Uppsala University in Uppsala, Sweden. Rat and mouse pancreatic islets and fetal porcine islet-like cell clusters (ICC) were prepared in Uppsala. All groups of islets were subsequently sent to the Department of Biotechnology, Norwegian Institute of Biotechnology, University of Trondheim, Trondheim, Norway. After 1 day in tissue culture, the islets were microencapsulated in alginate then cultured and sent back to Uppsala the next day. After either overnight culture (day 1) or 6 days of culture (day 6), the microencapsulated islets were examined for their insulin content and insulin release. Nonencapsulated islets from the same isolations were used as controls. RESULTS: The insulin content of rodent and human islets was not affected by microencapsulation, whereas porcine ICC showed a diminished insulin content. Microencapsulated porcine ICC also had a marked reduction in their insulin secretion in response to stimulation with glucose or glucose + theophylline both on days 1 and 6 in tissue culture. Mouse islets showed a reduced insulin response at both time points. Rat islets exhibited an inhibition of insulin secretion on day 1, but this had been restored by day 6. Human islets had well-preserved insulin secretion after both days 1 and 6. Microencapsulated human islets showed a normal morphology 3-4 weeks after intraperitoneal transplantation to nude mice. CONCLUSIONS: Pancreatic islets isolated from human, rat, and mouse donors show a glucose-stimulated insulin release in vitro after microencapsulation and repeated transports between laboratories. The insulin secretory capacity of microencapsulated human and rat islets was preserved best, whereas mouse islets and particularly fetal porcine ICC were impaired by microencapsulation.Journal ArticleResearch Support, Non-U.S. Gov'tinfo:eu-repo/semantics/publishe