MV-mimicking micelles loaded with PEG-serine-ACP nanoparticles to achieve biomimetic intra/extra fibrillar mineralization of collagen in vitro

Since their discovery, matrix vesicles (MVs) containing minerals have received considerable attention for their role in the mineralization of bone, dentin and calcified cartilage. Additionally, MVs' association with collagen fibrils, which serve as the scaffold for calcification in the organic matrix, has been repeatedly highlighted. The primary purpose of the present study was to establish a MVs–mimicking model (PEG-S-ACP/micelle) in vitro for studying the exact mechanism of MVs-mediated extra/intra fibrillar mineralization of collagen in vivo. In this study, high-concentration serine was used to stabilize the amorphous calcium phosphate (S-ACP), which was subsequently mixed with polyethylene glycol (PEG) to form PEG-S-ACP nanoparticles. The nanoparticles were loaded in the polysorbate 80 micelle through a micelle self-assembly process in an aqueous environment. This MVs–mimicking model is referred to as the PEG-S-ACP/micelle model. By adjusting the pH and surface tension of the PEG-S-ACP/micelle, two forms of minerals (crystalline mineral nodules and ACP nanoparticles) were released to achieve the extrafibrillar and intrafibrillar mineralization, respectively. This in vitro mineralization process reproduced the mineral nodules mediating in vivo extrafibrillar mineralization and provided key insights into a possible mechanism of biomineralization by which in vivo intrafibrillar mineralization could be induced by ACP nanoparticles released from MVs. Also, the PEG-S-ACP/micelle model provides a promising methodology to prepare mineralized collagen scaffolds for repairing bone defects in bone tissue engineering.     

Getting a grip on the intertidal: flow microhabitat and substratum type determine the dislodgement of the crab Pachygrapsus crassipes (Randall) on rocky shores and in estuaries

Hydrodynamic forces can affect survival as well as limit the movement of motile benthic animals. An animal's danger of dislodgement depends on the hydrodynamic forces it experiences in its microhabitat relative to the force required to dislodge it (tenacity) from the substratum. We measured water flow and substratum characteristics in two different habitats of the shore crab Pachygrapsus crassipes: a wave-swept rocky shore and an intertidal mudflat. The maximum water velocities and accelerations in the microhabitats of the crabs at the wave-swept site were three times and two times greater, respectively, than at the mudflat site. In the laboratory, we measured the tenacity of crabs of various sizes on different substrata, and also measured their drag, lift and added-mass coefficients. Using these data, we calculated the flow conditions under which crabs would be overturned or sheared off the substratum in their two habitats. The net horizontal force (drag plus acceleration reaction) required to dislodge a crab on a rugose rock substratum was an order of magnitude greater than on smooth rock and two orders of magnitude greater than on mud. Our calculations indicate that, under non-storm conditions, crabs will not be dislodged from the substratum in either the mudflat or the wave-swept habitat when grasping the substratum with maximum tenacity. Moving crabs have lower tenacity and our calculations predict that hydrodynamic forces will restrict the mobility of large crabs more than that of small ones on smooth, but not on rugose rock.     

An enzymic method for the determination of chitin and chitosan in fungal cell walls

The free and N-acetyl glucosamine contents, serving as a measure of the amounts of chitosan and chitin respectively, were determined in the chitinase hydrolysates of the cell wall of a wild strain ofNeurospora crassa. Chitinase, obtained from cultures ofSerratia marcescens, could hydrolyse the cell wall completely apart from being capable of hydrolysing preparations of chitin and chitosan. The free and N-acetyl glucosamines, released by chitinase hydrolysis, were determined by a modified Morgan-Elson reaction carried out in the presence and absence of acetic anhydride. The method is capable of estimating chitin and chitosan contents in as little as 100 μg of cell wall material.     

Membrane turnover in crab photoreceptors studied by high-resolution scanning electron microscopy and by a new technique of thick-section transmission electron microscopy

Summary Crab photoreceptors were examined after treatment by the osmium-DMSO-osmium method for high-resolution scanning electron microscopy. This technique of specimen preparation was also adapted for transmission electron microscopy, enabling sections up to 1 urn thick to be viewed in a conventional microscope at 75 kV. With appropriate pretreatment, some cytoskeletal elements can be visualised by both techniques. The methods were then used to investigate some of the daily changes known to occur in photoreceptor cell structure. Striking differences were found in the structure of Golgi bodies present in retinula cells during the synthesis and breakdown phases of the daily cycle of photoreceptor membrane turnover. Cyclic changes were also noticed in the mitochondria of retinula cells, and additional evidence was found for a previously proposed model of rhabdomeral microvillus formation.     

Immobilization of "Disguised" yeast in chemically crosslinked chitosan beads

A new simple method for the preparation of chemically crosslinked chitosan beads is presented. It consists of the dropwise addition of 2-3% (w/v) low molecular weight chitosan solution containing 2% (w/v) glyoxal in 1% (w/v) tetrasodiumdiphosphate, pH 8.0. Immobilized viable baker's yeast (Saccharomyces cerevisiae) could be obtained via gel entrapment within the new beads when means preventing their direct contact with soluble chitosan were provided, "disguising" the cells until gelation and crosslinking were completed. Such means included cell suspension in castor oil or mixing with carboxymethyl-cellulose powder. Application of these means was shown to be necessary, as cells exposed to soluble chitosan immediately lost their viability and glycolytic activity. Yeast disguised in castor oil was also protected from bead reinforcement by glutaraldehyde treatment, significantly strengthening bead stability while operating under acidic conditions. This capability was demonstrated by continuous ethanol production by chitosan entrapped yeast. (c) 1994 John Wiley & Sons, Inc.     

Regulation of circulating levels of the crustacean hyperglycemic hormone: evidence for a dual feedback control system

The effects of glutamate, aspartate, glycine, proline, alanine, taurine, glycerol, glucose and lactate injections on the haemolymph levels of the crustancean hyperglycemic hormone and/or glucose and lactate in the shore crab, Carcinus maenas, were investigated. Only glucose and lactate caused significant changes of hyperglycaemic hormone levels. Glucose injections resulted in a drop of both hormone and lactate, while lactate had an opposite effect, i.e. it raised both crustacean hormone and glucose levels. The results suggest that during increases in glycolytic flux, lactate may cause a release of hormone by a positive feedback mechanism. The hormone would then stimulate glycogenolysis, thus increasing glucose availability. If more glucose is released than is metabolized, excess glucose may leak from the cells and suppress crustancean hyperglycemic hormone release from the X-organ/sinus gland complex by negative feedback.Abbreviations ABTS 2,2-azino-bis (3-ethylbenzthiazoline sulphonic acid) - ANOVA one-way analysis of variance - BSA bovine serum albumin - BW body weight - CHH crustacean hyperglycemic hormone - ELISA cnzyme-liked immunosorbent assay - GIH gonadinhibiting hormone - IgG immunoglobin G - MIH moult-inhibiting hormone - MTGXO medulla terminalis X-organ - PB sodium phosphate buffer - PBS phosphate buffered saline - Pi inorganic phosphate - XO-SG X-organ-sinus gland complex     

Alginate-chitosan coacervation in production of artificial seeds

Survival of secondary embryoids of winter oilseed rape (Brassica napus ssp. oleifera cv. Primor) has been used as an assay for the development of artificial seeds involving complex coacervation of alginate (polyanion) with chitosan (polycation). Germination frequency of 100% was achieved for encapsulated embryoids when alginate formed the inner matrix and chitosan the outer layer. When the matrix makeup was reversed, there was no germination of embryoids. The artificial seeds produced were hardened in dilute alkaline solutions of NaOH and Ca(OH)(2). An optimum setting time could be selected based on a quantitative measurement of resistance of hardened capsules to compression and the germination frequency of the encapsulated embryoids. (c) 1993 John Wiley & Sons, Inc.     

Alginate/chitosan hydrogels as perspective transport systems for cefotaxime

This work reports synthesis of pH-responsive alginate/chitosan hydrogel spheres with the average diameter of 2.0 ± 0.05 mm, which contain cefotaxime that is an antibiotic of the cefalosporine group. The spheres provided the cefotaxime encapsulation efficiency of 95 ± 1%. An in vitro release of cefotaxime from the spheres in the media that simulate human biological fluids in peroral delivery conditions was found to be a pH-dependent process. The analysis of cefotaxime release kinetics by the Korsmeyer–Peppas model revealed a non-Fickian mechanism of its diffusion, which may be related to intermolecular interactions occurring between the antibiotic and chitosan. Conductometry, UV spectroscopy, and IR spectroscopy were used to study complexation of chitosan with cefotaxime in aqueous media with varied pH, characterize the composition of the complexes, and calculate their stability constants. The composition of the cefotaxime–chitosan complexes was found to correspond to the 1.0:4.0 and 1.0:2.0 molar ratios of the components at pH 2.0 and 5.6, respectively. Quantum chemical modeling was used to evaluate energy characteristics of chitosan–cefotaxime complexation considering the influence of a solvent.