Laura Treccani

Publications of Laura Treccani

• Osteoblast viability on hydroxyapatite with well-adjusted submicron and micron surface roughness as monitored by the proliferation reagent WST-1.

  Authors: Marzellus Große Holthaus, Laura Treccani, Kurosch Rezwan

  Journal of biomaterials applications. 01/2012;

  The impact of the cell surface roughness on titanium alloys used for biomedical implants has been extensively studied, whereas the dependency of human osteoblast viability on hydroxyapatite (HA)The impact of the cell surface roughness on titanium alloys used for biomedical implants has been extensively studied, whereas the dependency of human osteoblast viability on hydroxyapatite (HA) submicron and micron surface roughness has hitherto not yet been investigated in detail. Therefore, we investigate in this study the effect of HA substrates with different well-adjusted surface roughness on human osteoblast proliferation using the standard colorimetric reagent WST-1. By grinding, we obtained HA surfaces with six levels of well-defined surface roughness ranging from Sa = 3.36 µm down to 0.13 µm, resulting in hydrophilic contact angles from 11° to 27°. Energy dispersive X-ray spectroscopy, X-ray diffraction, and X-ray fluorescence measurements confirmed that neither grinding paper residues nor changes of the crystal structure were introduced to the HA substrates by the grinding process. By applying this simple surface treatment, we were able to isolate other effects from surface chemistry, crystal structure, and relative density. The changes of the osteoblast proliferation (WST-1 assay) on these different roughened HA surfaces after 7 days were found to be insignificant (p > 0.05), evaluated by one-way analysis of variance and Tuckey's Multiple Comparison Method. The results of this study show that all roughened HA surfaces, regardless of the microtopography, are biocompatible and allow osteoblast attachment, proliferation, and collagen type I production. The comparison with surface roughness used for standard Ti-based implants yielded that for HA no finishing process is necessary to ensure a sound human osteoblast cell proliferation in vitro.

• Fluorescence labeling of colloidal core-shell particles with defined isoelectric points for in vitro studies.

  Authors: Timo Daberkow, Fabian Meder, Laura Treccani, Marco Schowalter, Andreas Rosenauer, Kurosch Rezwan

  Acta biomaterialia. 11/2011; 8(2):720-7.

  In the light of in vitro nanotoxicological studies fluorescence labeling has become standard for particle localization within the cell environment. However, fluorescent labeling is also known toIn the light of in vitro nanotoxicological studies fluorescence labeling has become standard for particle localization within the cell environment. However, fluorescent labeling is also known to significantly alter the particle surface chemistry and therefore potentially affect the outcome of cell studies. Hence, fluorescent labeling is ideally carried out without changing, for example, the isoelectric point. A simple and straightforward method for obtaining fluorescently labeled spherical metal oxide particles with well-defined isoelectric points and a narrow size distribution is presented in this study. Spherical amorphous silica (SiO2, 161 nm diameter) particles were used as the substrate material and were coated with silica, alumina (Al2O3), titania (TiO2), or zirconia (ZrO2) using sol-gel chemistry. Fluorescent labeling was achieved by directly embedding rhodamine 6G dye in the coating matrix without affecting the isoelectric point of the metal oxide coatings. The coating quality was confirmed by high resolution transmission electron microscopy, energy filtered transmission electron microscopy and electrochemical characterization. The coatings were proven to be stable for at least 240 h under different pH conditions. The well-defined fluorescent particles can be directly used for biomedical investigations, e.g. elucidation of particle-cell interactions in vitro.

• Protein adsorption on colloidal alumina particles functionalized with amino, carboxyl, sulfonate and phosphate groups.

  Authors: Fabian Meder, Timo Daberkow, Laura Treccani, Michaela Wilhelm, Marco Schowalter, Andreas Rosenauer, Lutz Mädler, Kurosch Rezwan

  Acta biomaterialia. 09/2011; 8(3):1221-9.

  Colloidal oxide particles in biomedical or biotechnological applications immediately become coated with proteins of the biological medium, a process which is strongly influenced by the surfaceColloidal oxide particles in biomedical or biotechnological applications immediately become coated with proteins of the biological medium, a process which is strongly influenced by the surface characteristics of the particles. Fundamental correlations between surface characteristics and the, so far mainly uncontrollable, protein adsorption are still not clear. In this study the surface of colloidal alumina particles (d(50)=179 ± 8 nm) was systematically adjusted with NH(2), COOH, SO(3)H and PO(3)H(2) functional groups to investigate the influence on the adsorption of the three model proteins, bovine serum albumin (BSA), lysozyme (LSZ) and trypsin (TRY). The surface functionalization is characterized and discussed in detail with regard to the morphology, isoelectric point, zeta potential, hydrophilic/hydrophobic properties, functional group density and stability. Protein-particle interaction was then assessed by evaluating the amount of protein adsorbed and the zeta potentials of protein-particle conjugates. Protein adsorption was found to be influenced by the type of functional group as well as the expected electrostatic forces under the given experimental conditions. The level of protein adsorption might, hence, be specifically controlled by the type of surface functionalization. Possible adsorption modes of BSA, LSZ and TRY on the particles are suggested by considering the spatial surface potential distribution of the proteins calculated from the protein database file. The particles presented provide an excellent prerequisite for further investigation of fundamental particle-protein interactions and the design of functionally graded materials for biomedical and biotechnological applications, e.g. as drug carriers or for protein purification.

• Adsorption and reduction of glutathione disulfide on α-Al2O3 nanoparticles: experiments and modeling.

  Authors: Ralf Dringen, Yvonne Koehler, Ludmilla Derr, Giulia Tomba, Maike M Schmidt, Laura Treccani, Lucio Colombi Ciacchi, Kurosch Rezwan

  Langmuir : the ACS journal of surfaces and colloids. 06/2011; 27(15):9449-57.

  Glutathione disulfide (GSSG; γ-GluCysGly disulfide) was used as a physiologically relevant model molecule to investigate the fundamental adsorption mechanisms of polypeptides onto α-aluminaGlutathione disulfide (GSSG; γ-GluCysGly disulfide) was used as a physiologically relevant model molecule to investigate the fundamental adsorption mechanisms of polypeptides onto α-alumina nanoparticles. Its adsorption/desorption behavior was studied by enzymatic quantification of the bound GSSG combined with zeta potential measurements of the particles. The adsorption of GSSG to alumina nanoparticles was rapid, was prevented by alkaline pH, was reversed by increasing ionic strength, and followed a nearly ideal Langmuir isotherm with a standard Gibbs adsorption energy of -34.7 kJ/mol. Molecular dynamics simulations suggest that only one of the two glutathionyl moieties contained in GSSG binds stably to the nanoparticle surface. This was confirmed experimentally by the release of GSH from the bound GSSG upon reducing its disulfide bond with dithiothreitol. Our data indicate that electrostatic interactions via the carboxylate groups of one of the two glutathionyl moieties of GSSG are predominantly responsible for the binding of GSSG to the alumina surface. The results and conclusions presented here can provide a base for further experimental and modeling studies on the interactions of biomolecules with ceramic materials.

• Perlinhibin, a cysteine-, histidine-, and arginine-rich miniprotein from abalone (Haliotis laevigata) nacre, inhibits in vitro calcium carbonate crystallization.

  Authors: Karlheinz Mann, Frank Siedler, Laura Treccani, Fabian Heinemann, Monika Fritz

  Biophysical journal. 09/2007; 93(4):1246-54.

  We have isolated a 4.785 Da protein from the nacreous layer of the sea snail Haliotis laevigata (greenlip abalone) shell after demineralization with acetic acid. The sequence of 41 amino acids wasWe have isolated a 4.785 Da protein from the nacreous layer of the sea snail Haliotis laevigata (greenlip abalone) shell after demineralization with acetic acid. The sequence of 41 amino acids was determined by Edman degradation supported by mass spectrometry. The most abundant amino acids were cysteine (19.5%), histidine (17%), and arginine (14.6%). The positively charged amino acids were almost counterbalanced by negatively charged ones resulting in a calculated isoelectric point of 7.86. Atomic-force microscopy studies of the interaction of the protein with calcite surfaces in supersaturated calcium carbonate solution or calcium chloride solution showed that the protein bound specifically to calcite steps, inhibiting further crystal growth at these sites in carbonate solution and preventing crystal dissolution when carbonate was substituted with chloride. Therefore this protein was named perlinhibin. X-ray diffraction investigation of the crystal after atomic-force microscopy growth experiments showed that the formation of aragonite was induced on the calcite substrate around holes caused by perlinhibin crystal-growth inhibition. The strong interaction of the protein with calcium carbonate was also shown by vapor diffusion crystallization. In the presence of the protein, the crystal surfaces were covered with holes due to protein binding and local inhibition of crystal growth. In addition to perlinhibin, we isolated and sequenced a perlinhibin-related protein, indicating that perlinhibin may be a member of a family of closely related proteins.

• Perlwapin, an abalone nacre protein with three four-disulfide core (whey acidic protein) domains, inhibits the growth of calcium carbonate crystals.

  Authors: Laura Treccani, Karlheinz Mann, Fabian Heinemann, Monika Fritz

  Biophysical journal. 11/2006; 91(7):2601-8.

  We have isolated a new protein from the nacreous layer of the shell of the sea snail Haliotis laevigata (abalone). Amino acid sequence analysis showed the protein to consist of 134 amino acids and toWe have isolated a new protein from the nacreous layer of the shell of the sea snail Haliotis laevigata (abalone). Amino acid sequence analysis showed the protein to consist of 134 amino acids and to contain three sequence repeats of approximately 40 amino acids which were very similar to the well-known whey acidic protein domains of other proteins. The new protein was therefore named perlwapin. In addition to the major sequence, we identified several minor variants. Atomic force microscopy was used to explore the interaction of perlwapin with calcite crystals. Monomolecular layers of calcite crystals dissolve very slowly in deionized water and recrystallize in supersaturated calcium carbonate solution. When perlwapin was dissolved in the supersaturated calcium carbonate solution, growth of the crystal was inhibited immediately. Perlwapin molecules bound tightly to distinct step edges, preventing the crystal layers from growing. Using lower concentrations of perlwapin in a saturated calcium carbonate solution, we could distinguish native, active perlwapin molecules from denaturated ones. These observations showed that perlwapin can act as a growth inhibitor for calcium carbonate crystals in saturated calcium carbonate solution. The function of perlwapin in nacre growth may be to inhibit the growth of certain crystallographic planes in the mineral phase of the polymer/mineral composite nacre.

• Abalone nacre insoluble matrix induces growth of flat and oriented aragonite crystals.

  Authors: Fabian Heinemann, Laura Treccani, Monika Fritz

  Biochemical and biophysical research communications. 06/2006; 344(1):45-9.

  Mollusc shell formation takes place in a preformed extracellular matrix, composed of insoluble chitin, coated with proteins and dissolved macromolecules. The water-soluble matrix is known to have aMollusc shell formation takes place in a preformed extracellular matrix, composed of insoluble chitin, coated with proteins and dissolved macromolecules. The water-soluble matrix is known to have a strong influence on the growth of CaCO(3), whereas the role of the insoluble matrix on mineralization is unclear. Therefore, we mineralized the EDTA (ethylenediaminetetraacetic acid) insoluble organic matrix of abalone nacre with a modified double-diffusion set-up, where the diffusing solutions were constantly renewed. Control experiments were performed with cellulose and chitosan foils. The mineralized matrices/foils were analyzed with SEM. We show that the insoluble matrix of abalone nacre induces the growth of flat and roughly polygonal CaCO(3) crystals. In some of the experiments with the insoluble matrix, the growth of three-dimensional parallel sheets of densely packed platelets inside the insoluble matrix was observed. XRD on these samples revealed that they consist of oriented aragonite.

• Protein-Mineral Interaction of Purified Nacre Proteins with Calcium Carbonate Crystals

  Authors: Laura Treccani

  http://elib.suub.uni-bremen.de/diss/docs/00010453.pdf.

  Nacre, a fascinating biogenic material consisting of calcium carbonate and organic molecules, presents remarkable mechanical and chemical properties that far exceed those of the pure components.Nacre, a fascinating biogenic material consisting of calcium carbonate and organic molecules, presents remarkable mechanical and chemical properties that far exceed those of the pure components. Nacre is created by a self-organizing process guided by organic macromolecules. In particular proteins specifically control and regulate the nucleation and growth of the mineral phase, influencing the crystal morphology and polymorph selection.In this work new strategies to extract and purify proteins from the nacre of Haliotis laevigata in their native and functional state were developed and mechanisms of interaction between native nacre proteins with calcium carbonate in solution and calcium carbonate minerals were investigated. In particular the function of the intracrystalline proteins, a novel group of proteins characterized for the first time in nacre, and perlucin and perlinhibin, two already sequenced nacre proteins, were elucidated. It has been shown that each nacre protein presents a very specific and unique function in terms of calcium carbonate mineralization.