Preparation of monolithic silica-chitin composite under extreme biomimetic conditions.

Chitin is a widespread renewable biopolymer that is extensively distributed in the natural world. The high thermal stability of chitin provides an opportunity to develop novel inorganic-organic composites under hydrothermal synthesis conditions in vitro. For the first time, in this work we prepared monolithic silica-chitin composite under extreme biomimetic conditions (80°C and pH 1.5) using three dimensional chitinous matrices isolated from the marine sponge Aplysina cauliformis. The resulting material was studied using light and fluorescence microscopy, scanning electron microscopy, Fourier transform infrared spectroscopy. A mechanism for the silica-chitin interaction after exposure to these hydrothermal conditions is proposed and discussed.

Preparation of chitin-silica composites by in vitro silicification of two-dimensional Ianthella basta demosponge chitinous scaffolds under modified Stöber conditions.

Chitin is a biopolymer found in cell walls of various fungi and skeletal structures of numerous invertebrates. The occurrence of chitin within calcium- and silica-containing biominerals has inspired development of chitin-based hybrids and composites in vitro with specific physico-chemical and material properties. We show here for the first time that the two-dimensional α-chitin scaffolds isolated from the skeletons of marine demosponge Ianthella basta can be effectively silicified by the two-step method with the use of Stöber silica micro- and nanodispersions under Extreme Biomimetic conditions. The chitin-silica composites obtained at 120 °C were characterized by the presence of spherical SiO2 particles homogeneously distributed over the chitin fibers, which probably follows from the compatibility of Si-OH groups to the hydroxyl groups of chitin. The biocomposites obtained were characterized by various analytical techniques such as energy dispersive spectrometry, scanning electron microscopy, thermogravimetric/differential thermal analyses as well as X-ray photoelectron spectroscopy, Fourier transform infrared and Raman spectroscopy to determine possible interactions between silica and chitin molecule. The results presented proved that the character and course of the in vitro chitin silicification in Stöber dispersions depended considerably on the degree of hydrolysis of the SiO2 precursor.

Identification and first insights into the structure and biosynthesis of chitin from the freshwater sponge Spongilla lacustris.

This work demonstrates that chitin is an important structural component within the skeletal fibers of the freshwater sponge Spongilla lacustris. Using a variety of analytical techniques ((13)C solid state NMR, FT-IR, Raman, NEXAFS, ESI-MS, Morgan-Elson assay and Calcofluor White Staining); we show that this sponge chitin is much closer to α-chitin, known to be present in other animals, than to ß-chitin. Genetic analysis confirmed the presence of chitin synthases, which are described for the first time in a sponge. The presence of chitin in both marine (demosponges and hexactinellids) and freshwater sponges indicates that this important structural biopolymer was already present in their common ancestor.

First report on chitinous holdfast in sponges (Porifera).

A holdfast is a root- or basal plate-like structure of principal importance that anchors aquatic sessile organisms, including sponges, to hard substrates. There is to date little information about the nature and origin of sponges' holdfasts in both marine and freshwater environments. This work, to our knowledge, demonstrates for the first time that chitin is an important structural component within holdfasts of the endemic freshwater demosponge Lubomirskia baicalensis. Using a variety of techniques (near-edge X-ray absorption fine structure, Raman, electrospray ionization mas spectrometry, Morgan-Elson assay and Calcofluor White staining), we show that chitin from the sponge holdfast is much closer to α-chitin than to ß-chitin. Most of the three-dimensional fibrous skeleton of this sponge consists of spicule-containing proteinaceous spongin. Intriguingly, the chitinous holdfast is not spongin-based, and is ontogenetically the oldest part of the sponge body. Sequencing revealed the presence of four previously undescribed genes encoding chitin synthases in the L. baicalensis sponge. This discovery of chitin within freshwater sponge holdfasts highlights the novel and specific functions of this biopolymer within these ancient sessile invertebrates.

Extreme Biomimetics: formation of zirconium dioxide nanophase using chitinous scaffolds under hydrothermal conditions.

Chitinous scaffolds isolated from the skeleton of marine sponge Aplysina cauliformis were used as a template for the in vitro formation of zirconium dioxide nanophase from ammonium zirconium(iv) carbonate (AZC) under extreme conditions (150 °C). These novel zirconia-chitin based composites were prepared for the first time using hydrothermal synthesis, and were thoroughly characterized using a plethora of analytical methods. The thermostability of the chitinous 3D matrix makes it ideal for use in the hydrothermal synthesis of monoclinic nanostructured zirconium dioxide from precursors like AZC. These zirconium-chitin composites have a high potential for use in a broad range of applications ranging from synthetic catalysis to biocompatible materials for bone and dental repair. The synthetic methods presented in this work show an attractive route for producing monoclinic zirconium dioxide on a 3D biocompatible scaffold with ease.

Isolation and identification of chitin in the black coral Parantipathes larix (Anthozoa: Cnidaria).

Until now, there is a lack of knowledge about the presence of chitin in numerous representatives of corals (Cnidaria). However, investigations concerning the chitin-based skeletal organization in different coral taxa are significant from biochemical, structural, developmental, ecological and evolutionary points of view. In this paper, we present a thorough screening for the presence of chitin within the skeletal formations of a poorly investigated Mediterranean black coral, Parantipathes larix (Esper, 1792), as a typical representative of the Schizopathidae family. Using a wide array variety of techniques ((13)C solid state NMR, Fourier transform infrared (FTIR), Raman, NEXAFS, Morgan-Elson assay and Calcofluor White Staining), we unambiguously show for the first time that chitin is an important component within the skeletal stalks as well as pinnules of this coral.

Simple method for preparation of nanostructurally organized spines of sand dollar Scaphechinus mirabilis (Agassiz, 1863).

Unique skeletal formations of marine invertebrates, including representatives of Echinodermata, have the unique potential to serve as templates for bio-inspired materials chemistry, biomimetics, and materials science. The sand dollar Scaphechinus mirabilis (Agassiz, 1983) is widely distributed in the northwest of the Pacific Ocean from southern Japan to the Aleutian Islands. This animal is the main source of naphtochinone-based substances. These compounds have recently drawn medical attention for their use as cardiological and ophthalmological drugs. Unfortunately, after extraction of the naphtochinones, the residual skeletons and spines of the sand dollars were usually discarded. Here, we report the first method for the preparation of nanostructurally organized spines of S. mirabilis, using a simple enzymatic and hydrogen peroxide-based treatment. Application of this method opens the way for development of non-wasteful environmentally clean technology of sand dollars as well-known industrial marine invertebrates.

Carboxymethylation of the fibrillar collagen with respect to formation of hydroxyapatite.

Control over crystal growth by acidic matrix macromolecules is an important process in the formation of many mineralized tissues. Highly acidic macromolecules are postulated intermediates in tissue mineralization, because they sequester many calcium ions and occur in high concentrations at mineralizing foci in distantly related organisms. A prerequisite for biomineralization is the ability of cations like calcium to bind to proteins and to result in concert with appropriate anions like phosphates or carbonates in composite materials with bone-like properties. For this mineralization process the proteins have to be modified with respect to acidification. In this study we modified the protein collagen by carboxymethylation using glucuronic acid. Our experiments showed unambigously, that N(epsilon)-carboxymethyllysine is the major product of the in vitro nonenzymatic glycation reaction between glucuronic acid and collagen. We hypothesized that the function of biomimetically carboxymethylated collagen is to increase the local concentration of corresponding ions so that a critical nucleus of ions can be formed, leading to the formation of the mineral. Thus, the self-organization of HAP nanocrystals on and within collagen fibrils was intensified by carboxymethylation.

Modification of collagen in vitro with respect to formation of Nepsilon-carboxymethyllysine.

Developing new biopolymer-based materials with bio-identical properties is a significant challenge in modern science. One interesting route to this goal involves the biomineralization of collagen, a pre-structured and widely available protein, into a material with interesting properties. A prerequisite for biomineralization is the ability of cations (e.g., calcium) to bind to the protein and to result in concert with appropriate anions (e.g., phosphate) in composite material with e.g., bone-like properties. In order to increase the number of binding sites it is necessary to modify the protein prior to mineralization. For this glucuronic acid (GA) was used due to its carbonyl and carboxyl groups to derivatize proteinogenic amino groups transferring them into negatively charged carboxyl groups. Our experiments showed for the first time, that Nepsilon-carboxymethyllysine is the major product of in vitro non-enzymatic glycosylation of collagen by glucuronic acid. For an unequivocal determination of the reaction products, the lysine residues of collagen and of the model peptide were carboxymethylated through a reductive alkylation with glyoxalic acid and compared to the glucuronic acid derivatives. Beside their identical mass spectra the common structure elements could be confirmed with FTIR. Thus, in the context of matrix engineering, by producing Nepsilon-carboxymethyllysine, glucuronic acid offers a convenient way of introducing additional stable acidic groups into protein matrices.

First evidence of chitin as a component of the skeletal fibers of marine sponges. Part I. Verongidae (demospongia: Porifera).

The Porifera (sponges) are often regarded as the oldest, extant metazoan phylum, also bearing the ancestral stage for most features occurring in higher animals. The absence of chitin in sponges, except for the wall of peculiar resistance bodies produced by a highly derived fresh-water group, is puzzling, since it points out chitin to be an autapomorphy for a particular sponge family rather than the ancestral condition within the metazoan lineage. By investigating the internal proteinaceous (spongin) skeleton of two demosponges (Aplysina sp. and Verongula gigantea) using a wide array of techniques (Fourier transform infrared (FTIR), Raman, X-ray, Calcofluor White Staining, Immunolabeling, and chitinase test), we show that chitin is a component of the outermost layer (cuticle) of the skeletal fibers of these demosponges. FTIR and Raman spectra, as well as X-ray difractograms consistently revealed that sponge chitin is much closer to the alpha-chitin known from other animals than to beta-chitin. These findings support the view that the occurrence of a chitin-producing system is the ancestral condition in Metazoa, and that the alpha-chitin is the primitive form in animals.

Mineralization behaviour of collagen type I immobilized on different substrates.

Collagen type I as a robust fibre protein and main component of the extracellular matrix of most tissues is increasingly utilized for surface engineering of biomaterials using different immobilization methods. In the present work we studied the mineralization behaviour of fibrillar collagen type I in simulated body fluid as a measure for conformational changes caused by adsorptive immobilization or immobilization by partial incorporation into the anodic oxide layer on c.p.-titanium using microscopic and vibration spectroscopic methods. Adsorptive immobilization on highly oriented pyrolytic graphite (HOPG) and c.p.-titanium without collagen were used as references. In the initial phase (1-24 h) the kinetics of formation and the morphology of calcium phosphate phases (CPP) are strongly influenced both by the substrate and the immobilization method. Compared to HOPG both types of immobilization on titanium increasingly inhibit the formation of CPP. For longer times (30 d) these initial differences disappear-mineralization product on titanium, irrespective of the presence of collagen, is a mixture of amorphous calcium phosphate and octacalcium phosphate. Contrary to this the mineralization of HOPG substrates results in hydroxy apatite. This is discussed with respect to the conditions during the immobilization as well as the resulting interactions between substrate and immobilized collagen. It is shown that the mineralization process exhibits a high sensitivity with respect to conformational changes caused by these interactions. Possible cell biological relevance of these conformational changes is discussed.