Giuseppe Balsamo

Chitosan-mediated stimulation of macrophage function

According to the modern definition of biocompatibility, a biocompatible material need not be inert but be bioactive. A benign reactivity implies that the reactivity has to be appropriate for the intended use. Chitosan, a non-acetylated or partially deacetylated chitin (a linear homopolymer of beta (1-4)-linked N-acetylglucosamine) has been proposed as a biomaterial because of its apparent satisfactory biocompatibility. The present investigation demonstrates that chitosan has an in vitro stimulatory effect on both macrophage nitric oxide (NO) production and chemotaxis. The macrophage NO secretion is attributed to the N-acetylglucosamine unit of the chitosan molecule rather than to the glucosamine residue (28 and 15 microM NO respectively). Moreover, the immune stimulatory effect of chitosan was very specific since other glycosaminoglycans, such as N-acetyl-D-mannosamine and N-acetyl-D-galactosamine, had no effects on NO production (5 and 8 respectively). In vivo experiments strengthen this hypothesis. Transmission electron microscopy analysis identifies the presence of many leucocytes in the specimens after 14 d post-implantation, showing poor healing processes (i.e. fibroblast proliferation and collagen deposition) that characterize the tissue repair at this time in our animal model.

Calcite in the statoconia of amphibians: A detailed analysis in the frog Rana esculenta

SummaryThe otoconia of Amphibia, especially of the frog Rana esculenta, were studied by X-ray diffraction and scanning electron microscopy (SEM). The SEM studies showed that the membranous labyrinth of Amphibia contains two populations of crystals, which can easily be distinguished by their forms. The X-ray diffraction data indicated that these two populations consist of calcite and aragonite; the endolymphatic sac, the saccule and the lagena contain aragonite, whereas calcite is only found in the otolithic membrane of the utricle. The genetic and functional significance of the existence of two crystalline forms of calcium carbonate in the membranous labyrinth are discussed.

Scanning electron microscopic and X-ray diffraction studies of otoconia in the lizard Podarcis s. sicula

SummaryThe otoliths of embryos and young animals of the lizard Podarcis s. sicula were studied by X-ray diffraction and scanning electron microscopy. Two types of crystal that give different X-ray diffraction patterns were found in the membranous labyrinth of Podarcis. The crystals consist of calcite or aragonite and are easily distinguished by scanning electron microscopy because of their different morphology. The two calcium carbonate crystal forms are not mixed at random but are present in the embryo from the very beginning in specific sites. The endolymphatic sac contains aragonite crystals while the saccule contains calcite crystals adjacent to the wall, in addition to a preponderance of aragonite crystals. The utricle and lagena contain only calcite crystals. The presence of two crystal forms of calcium carbonate in the membranous labyrinth are discussed in terms of differing genetic and functional significance.

Calcification processes in the chick otoconia and calcium binding proteins : patterns of tetracycline incorporation and calbindin-D28K distribution

In order to clarify the otoconia formation and turnover, tetracycline, an antibiotic that precipitates at calcifying fronts and serves as a fluorescent marker, was injected into eggs at different stages of chick embryonic development, as well as into postnatal chicken and into adult animals. The changes in the intensity, location patterns and time course of fluorescent labelling in each examined stage in the otolithic organs was studied. The presence and distribution of calbindin (CB)-D28K, one of the calcium-binding proteins constantly found in the mammalian and chicken cochlea and also in otolithic membrane of some adult mammals, was studied. Results in embryonal stages, postnatal and adult animals allow us to postulate that otoliths are mainly produced during the embryonal phase, but they may also be produced throughout the whole life span. Results also indicate that otoconia are dynamic structures which undergo turnover. The correspondence between the patterns of CB-D28K immunoreactivity and tetracycline fluorescence may indicate that CB-D28K participates in the formation of otoconia.

Gentamicin ototoxicity in the saccule of the lizard Podarcis Sicula induces hair cell recovery and regeneration

There is little information available on the susceptibility of reptilian saccule hair cells to ototoxin-induced sensory damage. In this study, we report morphological evidence of hair cell recovery and regeneration after damage induced by gentamicin in the saccule of a lizard. We perform morphological analysis using scanning electron microscopy and confocal laser scanning microscopy with actin and calbindin as markers for hair cells and tubulin as a marker for supporting cells. The data were consistent: gentamicin induced damage in the hair cells, and the damage increased with increasing duration of treatment. Initially, the saccule appeared unhealthy. Subsequently, the sensory hair cells became compromised, with fused stereovilli, followed by widespread loss of hair cell bundles from the hair cells. Finally, numerous hair cells were lost. Morphologically, the saccule appeared normal 28days after gentamicin treatment. Using a mitogenic marker, we tested whether or not there is hair cell regeneration following administration of gentamicin. We found evidence of bromodeoxyuridine incorporation first in supporting cell nuclei and subsequently in hair cell nuclei. This indicates that a process of sensory epithelium repair and hair cell regeneration occurred, in both extrastriolar and striolar regions, and that the recovery was due to both the proliferation of supporting cells and, as seems likely, self-repair of hair cell bundles.

Localization of calbindin D-28K in the otoconia of lizard Podarcis sicula.

The membranous labyrinth of lizard Podarcis sicula contains calcite and aragonite crystals. Saccule, utricle and lagena contain calcite crystals while aragonite crystals are present only in the saccule where they are very abundant. We have recently demonstrated the presence of calbindin D-28K in the organic matrix of lizard P. sicula otoconia. In order to define its localization, since calbindin modulates cellular Ca2+ level, otoconia from utricle and lagena were collected separately from those from saccule and then otoconial proteins were extracted. Immunoblot assay on proteins extracted from the otoconia and confocal laser scanning microscope analyses of otoconia using monoclonal anti-calbindin D-28K antibodies indicated that calbindin D-28K is a protein typical of aragonite crystals.

Calbindin D28K is a component of the organic matrix of lizard Podarcis sicula otoconia

The factors controlling otoconia growth are not well known but it seems that the type of proteins contained in the otoconia regulates the initiation and/or the subsequent rates of crystal growth determining the morphology and the size of the final crystal. In order to clarify the mechanism of otoconia formation and their turnover, major proteins contained in the otoconia from the maculae of the saccule, utricle and lagena of inner ear of lizard Podarcis sicula were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Coomassie staining of SDS-PAGE resulted in a major broad band of 15 kDa and four other bands of 21, 28, 45 and 97 kDa. The proteins of 15, 21, 28 and 45 kDa were separated by high-pressure liquid chromatography on a C-4-reverse-phase column and the incubation of blots with monoclonal anti-Calbindin D28K antibodies indicated that the band of 28 kDa was Calbindin D28K, a calcium-binding protein.

Morphological and biochemical analyses of otoliths of the ice‐fish Chionodraco hamatus confirm a common origin with red‐blooded species

The morphology and composition of the three otoliths of the Antarctic ice‐fish Chionodraco hamatus were studied by scanning electron microscopy and X‐ray diffraction. The composition of the sagitta, lapillus and asteriscus protein matrices was also analysed by sodium dodecyl sulphate‐polyacrylamide gel electrophoresis, western blots and confocal laser scanning microscopy to reveal the presence of and to localize the calcium‐binding proteins calmodulin, calbindin and S‐100. Morphological results indicated that the otoliths in this ice‐fish were similar to those of Trematomus bernacchii, a red‐blooded Antarctic species [B. Avallone et al. (2003 ) J. Submicrosc. Cytol. Pathol. 35, 69–76], but rather different from those of other teleosts. These two Antarctic species possessed a completely vateritic asteriscus, whereas their sagitta and lapillus were made mostly of aragonite. Parallel analysis of protein patterns in C. hamatus and T. bernacchii revealed that the sagitta significantly differed from the lapillus and asteriscus in both species. The sagitta did not contain the S‐100 protein and showed calmodulin and calbindin located in discontinuous or incremental zones, respectively. These results demonstrate that the otoliths of C. hamatus and T. bernacchii share more resemblances than differences and support the idea of a common origin of these species.

Morphogenesis of otoliths during larval development in brook lamprey, Lampetra planeri

Otolith morphogenesis of the brook lamprey, Lampetra planeri, was analysed from larval to adult stages. The brook lamprey remains juvenile for about 4 years, facilitating analysis of otoliths maturation that permits to identify relevant evolutionary traits in this primitive species and to compare our results with more evoluted species of vertebrate taxa. We combined histochemical, immunohistochemical, scanning electron microscopy, elemental analysis and X‐ray diffraction of lamprey otoliths to establish possible relationships between otolithic mass, individual crystals, the otolithic organic substance that binds individual otoconia together and the inorganic elements that mineralize the lamprey otoliths. Histochemical analysis of the otoliths suggests that mineralization occurs gradually, beginning near the apex of the secretory epithelium. Then, the otoconia increase in size by deposition of layers of a dense crystalline substance. Immunohistochemical reactivity of calcium binding proteins indicates that calmodulin, calbindin, S‐100 and parvalbumin are parts of the uncalcified organic mass that holds otoconia together. Imaging of the immunoreactivity of each protein by Confocal Laser Scanning Microscopy in ammocoete at the first year of the larval stage shows weak reaction products which, however, gradually increase in intensity, with peak value in ammocoete at the fourth year of the larval stage.