René Born

Biomimetic coatings functionalized with adhesion peptides for dental implants.

A complete biological integration into the surrounding tissues (bone, gingiva) is a critical step for clinical success of a dental implant. In this work biomimetic coatings consisting either of collagen type I (for the gingiva region) and hydroxyapatite (HAP) or mineralized collagen (for the bone interface) have been developed as suitable surfaces regarding the interfaces. Additionally, using these biomimetic coatings as a matrix, adhesion peptides were bound to further increase the specificity of titanium implant surfaces. To enhance cell attachment in the gingiva region, a linear adhesion peptide developed from a laminin sequence (TWYKIAFQRNRK) was bound to collagen, whereas for the bone interface, a cyclic RGD peptide was bound to HAP and mineralized collagen using adequate anchor systems. The biological potential of these coatings deduced from cell attachment experiments with HaCaT human keratinocytes and MC3T3-E1 mouse osteoblasts showed the best results for collagen and laminin sequence coating for the gingiva region and mineralized collagen and RGD peptide coatings for regions with bone contact. Our concept opens promising approaches to improve the biological integration of dental implants.© 2001 Kluwer Academic Publishers

Nanostructural organization of naturally occurring composites-part II: silica-chitin-based biocomposites

Investigations of the micro-and nanostructures and chemical composition of the sponge skeletons as examples for natural structural biocomposites are of fundamental scientific relevance. Recently, we show that some demosponges (Verongula gigantea, Aplysina sp.) and glass sponges (Farrea occa, Euplectella aspergillum) possess chitin as a component of their skeletons. The main practical approach we used for chitin isolation was based on alkali treatment of corresponding external layers of spicules sponge material with the aim of obtaining alkali-resistant compounds for detailed analysis. Here, we present a detailed study of the structural and physicochemical properties of spicules of the glass sponge Rossella fibulata. The structural similarity of chitin derived from this sponge to invertebrate alpha chitin has been confirmed by us unambiguously using physicochemical and biochemical methods. This is the first report of a silica-chitin composite biomaterial found in Rossella species. Finally, the present work includes a discussion related to strategies for the practical application of silica-chitin-based composites as biomaterials.

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 β-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.

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.

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.

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 β-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.

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 Nɛ-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.

Demineralisation von natürlichen Silikat-basierten Biomaterialien: Neue Strategie zur Isolation organischer Gerüststrukturen

The objects of our study were anchoring spicules from the metre-long stalk of the glass rope sponge (Hyalonema sieboldi) which are remarkable for their size, durability, high flexibility and their exceptional fibre-optical properties which together have aroused interest in them as a novel biomimetic material. Sponge spicules are known to be hierarchically structured but the nature of the organic template on which silica is deposited has eluded identification because the hydrogen fluoride solutions generally used for desilicification drastically change the structure of proteins. To overcome this obstacle, we developed novel, slow-etching methods, which use solutions of 2.5 M NaOH at 37 °C and take 14 days. We show for the first time that the same class of proteins (collagen) involved in cartilage and bone formation also forms the matrix and deposition site of amorphous silica in sponge spicules. Our finding holds promise for development of new biomimetically fabricated, collagenbased, silica composites with applications in material science, biomedicine and other modern technologies. Acknowledgements We thank A. Shevchenko (MPI of Molecular Cell Biology and Genetics, Dresden) and his team for the protein identification study; C. Eckert, A. Ereskovsky, D. Krylova, U. Schwarzenbolz, M. Wollenweber, G. Richter, H. Meissner, A. Mensch, and T. Douglas for discussion and their technical assistance. Literatur [1] Aizenberg J., Weaver J. C., Thanawala M. S., Sundar V. C., Morse D. E., Fratzl P.: Skeleton of Euplectella sp.: Structural Hierarchy from the Nanoscale to the Macroscale. Science 309: 275–278, 2005 [2] Levi C., Barton J. L., Guillemet C., Le Bras E., Lehuede P.: A remarkably strong natural glassy rod: the anchoring spicule of the Monorhaphis sponge. J. Mater. Sci. Lett. 8: 337– 339, 1998 [3] Aizenberg J., Sundar V. C., Yablon A. D., Weaver J. C., Chen G.: Biological glass fibers: Correlation between optical and structural properties. Proc. Natl Acad. Sci USA 101 (10): 3358–3363, 2004 [4] Tabachnik K. R.: Adaptation of the Hexactinellid sponges to deep-sea life. Fossil and recent sponges. J. Reitner & H. Keupp. Berlin 1991 [5] Beaulieu S. E.: Life in glass houses: sponge stalk communities in the deep sea. Mar. Biol. 138: 803–817, 2001 [6] Müller W. E. G., Schwertner H. & Müller I.: Porifera a reference phylum for evolution and bioprospecting: the power of marine genomics. Keio. J. Med. 53 (3): 159–165, 2004 [7] Lopez P. J., Gautier C., Livage J. & Coradin T.: Mimiking Biogenic Silica Nanostructures Formation. Curr. Nanosci. 1: 73–78, 2005 [8] Uriz M-J., Turon X., Becerro M. A., Agell G.: Siliceous Spicules and Skeleton Frameworks in Sponges: Origin, Diversity, Ultrastructural Patterns, and Biological Functions. Microsc. Res. Tech. 62: 279–299, 2003 [9] Weaver J. C., Morse D. E.: Molecular Biology of Demosponge Axial Filaments and Their Roles in Biosilification. Microsc. Res. Tech. 62: 356–367, 2003 [10] Kölliker A.: Der feinere Bau der Protozoen. Wilhelm Engelmann Leipzig 1864 [11] Sollas W. J.: Report on the Tetractinellida collected by H.M.S. Challenger during the years 1873-76. In: Thomson, C.W., Murray, J. (eds.) Report on the scientific results of the ARBEITEN ORIGINAL Hermann Ehrlich: Demineralisation von natürlichen Silikat-basierten Biomaterialien

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.