Marcos Farina

Characterization of a bovine collagen–hydroxyapatite composite scaffold for bone tissue engineering

Different biomaterials have been used as scaffolds for bone tissue engineering. Here we characterize a biomaterial composed of sintered (1100 degrees C) and powdered hydroxyapatite (HA) and type I collagen (Coll), both of bovine origin, designed for osteoconductive and osteoinductive scaffolds. Coll/HA proportions were 1/2.6 and 1/1 (wet weight), and particles sizes varied from 200 to 400 microm. Vv (volume density) and Sv (surface to volume density) for the HA particles in the composite ranged from 0.48 +/- 0.06 to 0.55 +/- 0.02 and 5.090 +/- 0.545 to 6.366 +/- 0.289 microm(-1), respectively. Due to the relatively small changes in Vv and Sv, a macroporosity could be characterized for the biocomposite. X-ray diffraction and infrared spectroscopy showed that the sintered bone was composed essentially of HA with minimum additional groups such as surface calcium hydroxide, surface and crystal water, free carbon dioxide and possibly brushite. Mass spectrometry detected carbonates at A and B sites of HA, and weakly bound to the structure. Human osteoblasts adhered and spread on both the HA particle surface and the collagen fibers, which seemed to guide cells between adjacent particles. The biocomposite studied has several characteristics considered as ideal for its use as a scaffold for osteoconduction and osteoinduction.

Rhodolith Beds Are Major CaCO3 Bio-Factories in the Tropical South West Atlantic

Rhodoliths are nodules of non-geniculate coralline algae that occur in shallow waters (<150 m depth) subjected to episodic disturbance. Rhodolith beds stand with kelp beds, seagrass meadows, and coralline algal reefs as one of the worlds four largest macrophyte-dominated benthic communities. Geographic distribution of rhodolith beds is discontinuous, with large concentrations off Japan, Australia and the Gulf of California, as well as in the Mediterranean, North Atlantic, eastern Caribbean and Brazil. Although there are major gaps in terms of seabed habitat mapping, the largest rhodolith beds are purported to occur off Brazil, where these communities are recorded across a wide latitudinal range (2°N - 27°S). To quantify their extent, we carried out an inter-reefal seabed habitat survey on the Abrolhos Shelf (16°50′ - 19°45′S) off eastern Brazil, and confirmed the most expansive and contiguous rhodolith bed in the world, covering about 20,900 km2. Distribution, extent, composition and structure of this bed were assessed with side scan sonar, remotely operated vehicles, and SCUBA. The mean rate of CaCO3 production was estimated from in situ growth assays at 1.07 kg m−2 yr−1, with a total production rate of 0.025 Gt yr−1, comparable to those of the worlds largest biogenic CaCO3 deposits. These gigantic rhodolith beds, of areal extent equivalent to the Great Barrier Reef, Australia, are a critical, yet poorly understood component of the tropical South Atlantic Ocean. Based on the relatively high vulnerability of coralline algae to ocean acidification, these beds are likely to experience a profound restructuring in the coming decades.

Phylogenetic affiliation and ultrastructure of uncultured magnetic bacteria with unusually large magnetosomes

Abstract Natural enrichments of magnetic bacteria from the Itaipu lagoon near Rio de Janeiro were dominated by coccoid-to-ovoid morphotypes that produced unusually large magnetosomes. To determine the phylogenetic position of these unusual microorganisms, 16S rRNA genes were retrieved from bacteria magnetically separated from sediment of the Itaipu lagoon by in vitro amplification and cloning of PCR products into a plasmid vector. Partial sequencing of the obtained clones revealed two clusters of closely related sequences affiliated to a distinct lineage consisting exclusively of magnetic bacteria within the α-subclass of Proteobacteria. For a detailed phylogenetic analysis, several almost complete sequences of the 16S rRNA genes were determined. One representative clone of each cluster provided a PCR template for the in vitro transcription of group-specific polynucleotide probes complementary to a variable region of the 16S rRNA molecule. At least three different morphotypes of magnetic bacteria were reliably identified by post-embedding hybridization of ultra-thin sections. Electron microscopic analyses of hybridized cells enabled for the first time a detailed description of the morphological variety and ultrastructure of phylogenetically identified, uncultured magnetic bacteria. Two distinct coccoid bacteria were identified by the transcript probe complementary to the 16S rRNA sequence mabrj12, whereas the probe complementary to the sequence mabrj58 allowed the identification of an ovoid morphotype that displayed magnetosomes with the largest volumes observed to date.

Effects of copper on Enteromorpha flexuosa (Chlorophyta) in vitro.

Enteromorpha species are widely used as biomonitors of copper (Cu) contamination in coastal waters, but the effects of Cu at the subcellular level and possible mechanisms of metal resistance are scarcely known. To contribute to the understanding of the Cu accumulation process in macroalgae species, we exposed adult individuals of Enteromorpha flexuosa to 50, 250, and 500 microg Cu/L in seawater for 5 days under controlled conditions. Thereafter, gross photosynthesis rate (GPR) and metal accumulation were measured. Conventional transmission electron microscopy (CTEM) and energy-dispersive X-ray analysis (EDXA) were used to study the metal accumulation process at the cellular level. Treatments with 250 and 500 microg Cu/L were observed to cause an inhibition of the GPR in the algae. Cu accumulation in samples exposed to 500 microgCu/L was 5284+/-561 microg Cu/g (dry wt), whereas in control samples (no Cu addition), accumulation was 9+/-1 microg/g. In cells of plants undergoing the 50 microg Cu/L treatment, the cytoplasm was clearly vacuolated. Thickening of cell walls and increase of relative number of starch granules and of lipid bodies were the main cellular changes observed in plants exposed to 250 and 500 microg Cu/L. EDXA of algae cells after 250 and 500 microg Cu/L exposure detected Cu mainly in vacuole precipitates. Cu was also detected in chloroplasts and in some epiphytic bacteria. It was concluded that E. flexuosa did not avoid penetration of Cu into the cytoplasm and consequently its toxic effects in concentrations of 250 and 500 microg Cu/L. Precipitates containing Cu in vacuoles should be related to a metal immobilization mechanism, minimizing the Cu toxicity for cells. The epiphytic bacteria can act as a biofilter diminishing the availability of free Cu(+2) for algae accumulation.

Microcapsules of alginate/chitosan containing magnetic nanoparticles for controlled release of insulin

The challenge of this work was to investigate the potential of alginate/chitosan beads containing magnetite nanoparticles as a drug delivery system. The insulin beads were prepared by dripping a solution of sodium alginate containing insulin into a CaCl(2) solution. Magnetite nanoparticles of 5 nm mean size were synthesized inside the alginate egg-box structure by co-precipitation of Fe(III) and Fe(II) in the presence of NH(4)OH. Quantitative analysis revealed that insulin encapsulation depends on the initial protein content and 35% of insulin was entrapped by alginate beads for a protein concentration of 10 wt%. It was verified that approximately 50% of the insulin was released to Milli-Q water in 800 h release experiments. The application of oscillating magnetic field increased three fold the insulin release. The results suggest that the alginate/chitosan system containing magnetite nanoparticles is a promising system for clinical applications of controlled release of insulin in the presence of an oscillating magnetic field in a subcutaneous implant approach.

Chemical and topographical influence of hydroxyapatite and β-tricalcium phosphate surfaces on human osteoblastic cell behavior

The objective of this work was to evaluate the relative role of the calcium phosphate surface chemistry and surface topography on human osteoblast behavior. Highly dense phosphate ceramics (single-phase hydroxyapatite HA and beta-tricalcium phosphates TCP) presenting two distinct nano roughnesses were produced. Some samples were gold-sputter coated in order to conveniently mask the surface chemical effects (without modification of the original roughness) and to study the isolated effect of surface topography on cellular behavior. Our results indicated that the nanotopography of the studied ceramics had no effect on the cellular adhesion (cell spreading, focal contacts and stress fibers formation). On the contrary, strong topographical effects were verified on cell proliferation and differentiation. Moreover, the phosphate chemistry was responsible for changes in adhesion, proliferation and cell differentiation. On TCP, it was shown that the main influent parameter was surface chemistry, which negatively affected the initial cell adhesion but positively affected the subsequent stage of proliferation and differentiation. On HA, the main influent parameter was surface topography, which increased cell differentiation but lowered proliferation.

Brown algae species as biomonitors of Zn and Cd at Sepetiba Bay, Rio de Janeiro, Brazil

Abstract In order to contribute to monitoring heavy metal contamination of Sepetiba Bay, Rio de Janeiro, Brazil, long term evaluation of Zn and Cd concentration was performed in two brown algae species, Padina gymnospora and Sargassum stenophyllum. In relation to Sepetiba Bay macroalgae community, these species were the most abundant in substrate cover. The algae metal concentration variation from 1990 to 1997 should be related to the inputs of metals released into the bay by industrial process of Zn production in the area. In situ uptake and release transplant experiments with Padina between Sepetiba Bay and a near uncontaminated area showed that the species could reflect the variation in metal environment availability. A lower reduction in metal concentration by plants transplanted to the uncontaminated area was observed. Analytical electron microscopy showed that the Zn in P. gymnospora from Sepetiba Bay was present as small deposits distributed in the cell walls.

Magnetic Microstructure of Bacterial Magnetite by Electron Holography

A brackish lagoon at Itaipu, Brazil, contains magnetotactic bacteria with unusually large magnetite magnetosomes (lengths 100–200 nm). The micromagnetic structures of the magnetosomes from two different coccoid organisms from the lagoon have been determined by electron holography. The results are consistent with single-magnetic-domain structure in the elongated magnetosomes from one organism and metastable, single-magnetic-domain structure in the larger, more equi-axed, magnetosomes from the other organism. The results are consistent with theoretical predictions of the transition dimension between stable and metastable single-domain structure in magnetite.

Simple homemade apparatus for harvesting uncultured magnetotactic microorganisms

Descrevemos um aparato simples para a captura de microrganismos magnetotacticos nao cultivados. Este aparato consiste em um recipiente de vidro com duas aberturas. Uma abertura maior na parte superior e usada para introduzir o sedimento e a agua. O sedimento e a agua sao previamente armazenados em um recipiente semitampado, previamente testado para a presenca de bacterias magnetotacticas. O aparato e exposto a um campo magnetico, devidamente alinhado, em uma bobina feita a mao e as bacterias sao removidas pela extremidade capilar da segunda abertura do recipiente. As bacterias coletadas podem entao ser usadas em estudos ultraestruturais usando a tecnica de imagem espectroscopica eletronica. Um grande numero de bacterias consistindo de cocos e bastonetes foi eficientemente coletado de diferentes ambientes. Este aparato e util para estudos microbiologicos sobre microrganismos magnetotacticos nao cultivaveis, especialmente em abordagens moleculares para investigacoes filogeneticas que fornecem informacoes sobre a diversidade natural de comunidades microbianas.

Magnetite (Fe3O4) and Greigite (Fe3S4) Crystals in Multicellular Magnetotactic Prokaryotes

Magnetotactic bacteria produce iron oxides, iron sulfides or both in organelles called magnetosomes. Most of these bacteria are unicellular and biomineralize magnetite (Fe3O4). In contrast, multicellular magnetotactic prokaryotes (MMPs) consisting of several gram-negative cells have only been known to crystallize the magnetic iron sulfide greigite (Fe3S4). In this work, we describe MMPs that mineralize magnetite in bullet-shaped crystals. Another unusual aspect is that magnetite occurs either as the only crystals or together with greigite crystals. MMPs containing only greigite in the magnetosomes occur in the same environment. These findings show that morphology, ultrastructure, and behavior are the main characteristics of the MMPs, not the type of magnetic crystal biomineralized in the magnetosomes.