Rapid prototyping of chitosan-coated alginate scaffolds through the use of a 3D fiber deposition technique.

Three dimensional, periodic scaffolds of chitosan-coated alginate are fabricated in a layer-by-layer fashion by rapid prototyping. A novel dispensing system based on two coaxial needles delivers simultaneously alginate and calcium chloride solutions permitting the direct deposition of alginate fibers according to any designed pattern. Coating of the alginate fiber with chitosan and subsequent cross-linking with EDC and genipin assured the endurance of the scaffold in the culture environment for a prolonged period of time. The cross-linking protocol adopted imparted to the scaffold a hierarchical chemical structure as evidenced by Confocal Laser Microscopy and FTIR spectroscopy. The core of the fibers making up the scaffold is represented by alginate chains cross-linked by ester bonds only, the periphery of the fiber is constituted by an inter-polyelectrolyte complex of alginate and chitosan cross-linked in all pair combinations. Fibers belonging to adjacent layers are glued together by the chitosan coating. Mechanical behavior of the scaffolds characterized by different layouts of deposition was determined revealing anisotropic properties. The biocompatibility and capability of the scaffolds to sustain hepatocyte (HepaRG) cultures were demonstrated. Typical hepatic functions such as albumin and urea secretion and induction of CYP3A4 enzyme activity following drug administration were excellent, thus proving the potential of these constructs in monitoring the liver specific function.

Zinc deficiency induces membrane barrier damage and increases neutrophil transmigration in Caco-2 cells.

Zinc may contribute to the host defense by maintaining the membrane barrier. In this study, we questioned whether zinc deficiency affects the membrane function and junctional structure of intestinal epithelial cells, causing increased neutrophil migration. We used the Caco-2 cell line grown in control (C), zinc-deficient, or zinc-replete medium until differentiation. Zinc deprivation induced a decrease of transepithelial electrical resistance and alterations to tight and adherens junctions, with delocalization of zonula occludens (ZO-1), occludin, beta-catenin, and E-cadherin. Disorganization of F-actin and beta-tubulin was also found in zinc deficiency. These changes were associated with a loss of the amounts of ZO-1, occluding, and beta-tubulin. In addition, zinc deficiency caused a dephosphorylation of occludin and hyperphosphorylation of beta-catenin and ZO-1. Disruption of membrane barrier integrity led to increased migration of neutrophils. In addition, zinc deficiency induced an increase in the secretion of interleukin-8, epithelial neutrophil activating peptide-78, and growth-regulated oncogene-alpha, alterations that were not found when culture medium was replete with zinc. These results provide new information on the critical role played by dietary zinc in the maintenance of membrane barrier integrity and in controlling inflammatory cell infiltration.

Enzymatic cross-linking versus radical polymerization in the preparation of gelatin PolyHIPEs and their performance as scaffolds in the culture of hepatocytes.

Highly open porous biodegradable scaffolds, based on gelatin A3, were fabricated with the aim of using them for tissue-engineering applications. The fabrication process is based on an emulsion-templating technique. In the preparation of gelatin scaffolds two different cross-linking procedures were adopted: (I) radical polymerization of the methacrylate functionalities, previously introduced onto the gelatin chains and (II) formation of isopeptide bridges among the gelatin chains promoted by the enzyme microbial transglutaminase. The method of cross-linking exerts a pronounced effect on the morphology of the porous biomaterials: radical polymerization of methacrylated gelatin allowed the production of scaffolds with a better defined porous structure, while the enzymatically cross-linked scaffolds were characterized by a thinner skeletal framework. A suitable sample of each kind of the differently cross-linked porous biomaterials was tested for the culture of hepatocytes. The scaffold obtained by radical polymerization possessed a morphology characterized by relatively large voids and interconnects, and as a consequence, it was more suitable for hepatocytes colonization. On the other hand, the enzymatically cross-linked scaffold resulted in less cytotoxicity and the cultured hepatocytes expressed a better differentiated phenotype, as evidenced by a greater expression and more correct localization of key adhesion proteins.

Monoamine oxidase A and B activities in embryonic chick hepatocytes: differential regulation by retinoic acid.

Monoamine oxidases (MAOs) A and B are two isoenzymes involved in the degradation of many biological amines in the nervous system and in peripheral organs. In the present work hepatocytes isolated from 14-day-old chick embryos were used as a model system to determine whether retinoic acid (RA) is capable of modulating the activity of the two MAO forms. RA is a retinoid that, by binding with nuclear receptors, interferes with the expression of specific genes in many differentiation processes. Enzymic activity was measured with a radiochemical method using serotonin and beta-phenylethylamine as preferential substrates for MAO A and MAO B, respectively. The specific activity of the two forms was measured in hepatocytes cultured for 24, 48 and 72 h in the presence and the absence of serum. RA stimulated MAO B but not MAO A activity, in a dose- and time-dependent way, and only in the presence of serum. Maximum stimulation (about 3.5-fold) was obtained after treatment with 5 microM RA for 72 h. Kinetic analysis of MAO B activity showed an increase in V(max) in treated hepatocytes (5 microM RA for 72 h) with no change in K(m). In conclusion, the present work shows that RA selectively elicits MAO B activity in cultured chick embryonic hepatocytes, this stimulation requires the presence of some factors present in the serum and is probably due to an increase in the number of enzyme molecules.