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Biomaterials (v.28, #30)

Editorial board (pp. ifc).

A single-lens polarographic measurement of oxygen permeability ( Dk) for hypertransmissible soft contact lenses by Mahendra Chhabra; John M. Prausnitz; Clayton J. Radke (pp. 4331-4342).
A novel polarographic apparatus is described that requires only a single soft contact lens (SCL) to ascertain oxygen permeabilities of hypertransmissible lenses. Unlike conventional methods where a range of lens thickness is needed for determining oxygen permeabilities of SCLs, the apparatus described here requires only a single-lens thickness. This is accomplished by minimizing (or completely eliminating) edge effects, boundary-layer resistances, and lens desiccation in the polarographic apparatus. By taking these effects into account, we measure reliable oxygen permeabilities of hypertransmissible SCLs (i.e., above 100 barrer). Results are reported for nine commercial SCLs ranging in permeability from 9 to 180 barrer. Measured single-lens oxygen permeabilities are in excellent agreement with those claimed by commercial manufacturers. Our new single-lens permeameter provides a reliable, efficient, and economical method for measuring oxygen permeabilities of commercial SCLs. The single-lens method offers a potential international standard for measuring oxygen permeabilities of SCLs up to 250 barrer.

Keywords: Soft contact lens; Oxygen permeability; Polarographic method; Single-lens technique; Mass-transfer resistance; Hypertransmissible

In vitro biocompatibility of an ultrafine grained zirconium by Saldana Laura Saldaña; Antonio Méndez-Vilas; Ling Jiang; Marta Multigner; Jose L. González-Carrasco; María T. Pérez-Prado; María L. González-Martín; Luis Munuera; Nuria Vilaboa (pp. 4343-4354).
We have investigated a novel ultrafine grained (UFG) Zr obtained by severe plastic deformation (SPD) which resulted in a refinement of the grain size by several orders of magnitude. Compared to conventional Zr, higher hardness values were measured on UFG Zr. Polished surfaces having similar topographical features from both materials were prepared, as assessed by atomic force microscopy (AFM). Surface hydrophobicity of Zr, evaluated by measuring water contact angles, was unaffected by grain size reduction. In vitro biocompatibility was addressed on conventional and UFG Zr surfaces and, for comparative purposes, a polished Ti6Al4V alloy was also investigated. Cell attachment and spreading, actin and β-tubulin cytoskeleton reorganisation, fibronectin secretion and cellular distribution as well as cell viability were evaluated by culturing human osteoblastic Saos-2 cells on the surfaces. The osteoblastic response to conventional Zr was found to be essentially identical to Ti6Al4V and was not affected by grain size reduction. In order to evaluate the ability of the surfaces to promote osteogenic maturation and bone matrix mineralisation, human mesenchymal cells from bone marrow were switched to the osteoblastic phenotype by incubation in osteogenic induction media. Compared to undifferentiated mesenchymal cells, alkaline phosphatase activity and formation of mineralisation nodules were enhanced to the same extent on both Zr surfaces and Ti6Al4V alloy after induction of osteoblastic differentiation. In summary, improved mechanical properties together with excellent in vitro biocompatibility make UFG Zr a promising biomaterial for surgical implants.

Keywords: Biocompatibility; Osteoblast; Mesenchymal stem cell; Nanotopography

Competitive-protein adsorption in contact activation of blood factor XII by Rui Zhuo; Christopher A. Siedlecki; Erwin A. Vogler (pp. 4355-4369).
Contact activation of blood factor XII (FXII, Hageman factor) is moderated by the protein composition of the fluid phase in which FXII is dissolved. Solution yield of FXIIa arising from FXII contact with hydrophilic activating particles (fully water-wettable glass) suspended in a protein cocktail is shown to be significantly greater than that obtained under corresponding activation conditions in buffer solutions containing only FXII. By contrast, solution yield of FXIIa arising from FXII contact with hydrophobic particles (silanized glass) suspended in protein cocktail is sharply lower than that obtained in buffer. This confirms that contact activation is not specific to anionic hydrophilic surfaces as proposed by the accepted biochemistry of surface activation. Rather, contact activation in the presence of proteins unrelated to the plasma coagulation cascade leads to an apparent specificity for hydrophilic surfaces that is actually due to a relative diminution of activation at hydrophobic surfaces and an enhancement at hydrophilic surfaces. Furthermore, the rate of FXIIa accumulation in whole-plasma and buffer solution is found to decrease with time in the continuous presence of activating surfaces, leading to a steady-state FXIIa yield dependent on the initial FXII solution concentration for both hydrophilic and hydrophobic procoagulant particles suspended in either plasma, protein cocktail, or buffer. These results strongly suggest that activation competes with an autoinhibition reaction in which FXIIa itself inhibits FXII→FXIIa. Experimental results are modeled using a reaction scheme invoking FXII activation and autoinhibition linked to protein adsorption to procoagulant surfaces, where FXII activation is presumed to proceed by either autoactivation (FXII⟶surfaceFXIIa) and autohydrolysis (FXII⟶FXIIa2FXIIa) in buffer solution or autoactivation and reciprocal activation (kallikrein-mediated hydrolysis) in plasma. FXII adsorption competition with other proteins in the fluid phase is proposed to affect the balance of activation and autoinhibition, leading to the observed moderation of FXIIa yield.

Keywords: Blood coagulation; FXII; Hageman factor; Contact activation; Autoactivation; Protein adsorption

Noninvasive nuclear factor- κB bioluminescence imaging for the assessment of host–biomaterial interaction in transgenic mice by T.-Y. Tin-Yun Ho; Y.-S. Yueh-Sheng Chen; C.-Y. Chien-Yun Hsiang (pp. 4370-4377).
The inflammatory response is a key component in the biocompatibility of biomaterials. Among the factors that control the development of inflammation is a critical molecule nuclear factor- κB (NF- κB). Therefore, the aim of this study was to assess the feasibility of noninvasive whole-body real-time imaging for the evaluation of host–biomaterial interaction in the NF- κB transgenic mice. Transgenic mice, carrying the luciferase gene under the control of NF- κB, were constructed. In vivo bioluminescence imaging showed that the constitutive and induced NF- κB activities of transgenic mice were detected in most of the lymphoid tissues, demonstrating that NF- κB-driven luminescence reflected the inflammatory response in vivo. By the implantation of genipin-cross-linked gelatin conduit (GGC) and bacterial endotoxin-immersed GGC in the dorsal region, we detected a strong and specific luminescent signal from the tissue around the bacterial endotoxin-immersed GGC implant. Histological and immunohistochemical analysis also demonstrated that inflammation, characterized by the infiltration of immune cells, the accumulation of fluid, and the activation of NF- κB, was evoked around the same region. The correlation between the bioluminescence imaging and histological changes indicated that noninvasive imaging technique could be used to monitor the real-time inflammation in the implanted mice.

Keywords: Inflammation; Bioluminescence imaging; Genipin-cross-linked gelatin conduit; Nuclear factor-; κ; B; Transgenic mice

Elastin as a biomaterial for tissue engineering by W.F. Daamen; J.H. Veerkamp; J.C.M. van Hest; T.H. van Kuppevelt (pp. 4378-4398).
Biomaterials based upon elastin and elastin-derived molecules are increasingly investigated for their application in tissue engineering. This interest is fuelled by the remarkable properties of this structural protein, such as elasticity, self-assembly, long-term stability, and biological activity. Elastin can be applied in biomaterials in various forms, including insoluble elastin fibres, hydrolysed soluble elastin, recombinant tropoelastin (fragments), repeats of synthetic peptide sequences and as block copolymers of elastin, possibly in combination with other (bio)polymers. In this review, the properties of various elastin-based materials will be discussed, and their current and future applications evaluated.

Keywords: Elastin; Elastic fibre; Biomaterial; Review

Structural and rheological characterization of hyaluronic acid-based scaffolds for adipose tissue engineering by Assunta Borzacchiello; Laura Mayol; Piera A. Ramires; Andrea Pastorello; Chiara Di Bartolo; Luigi Ambrosio; Evelina Milella (pp. 4399-4408).
In this study the attention has been focused on the ester derivative of hyaluronic acid (HA), HYAFF®11, as a potential three-dimensional scaffold in adipose tissue engineering. Different HYAFF®11 sponges having different pore sizes, coated or not coated with HA, have been studied from a rheological and morphological point of view in order to correlate their structure to the macroscopic and degradation properties both in vitro and in vivo, using rat model. The in vitro results indicate that the HYAFF®11 sponges possess proper structural and mechanical properties to be used as scaffolds for adipose tissue engineering and, among all the analysed samples, uncoated HYAFF®11 large-pore sponges showed a longer lasting mechanical stability. From the in vivo results, it was observed that the elastic modulus of scaffolds seeded with preadipocytes, the biohybrid constructs, and explanted after 3 months of implantation in autologous rat model are over one order of magnitude higher than the corresponding values for the native tissue. These results could suggest that the implanted scaffolds can be invaded and populated by different cells, not only adipocytes, that can produce new matrix having different properties from that of adipose tissue.

Keywords: Adipose tissue engineering; Hyaluronic acid; Scaffold; Viscoelasticity

Engineering RGD nanopatterned hydrogels to control preosteoblast behavior: A combined computational and experimental approach by Wendy A. Comisar; Nikolas H. Kazmers; David J. Mooney; Jennifer J. Linderman (pp. 4409-4417).
The adhesion ligand arginine–glycine–aspartic acid (RGD) has been coupled to various materials to be used as tissue culture matrices or cell transplantation vehicles, and recent studies indicate that nanopatterning RGD into high-density islands alters key cell behaviors. Previous studies have failed, however, to conclusively decouple the effects of RGD bulk density and individual pattern parameters (i.e. RGDs/island and island distribution) on these altered cell responses. Using a nanopatterned RGD-coupled alginate hydrogel matrix, this work combines computational, statistical and experimental approaches to elucidate the effects of RGD patterns on four key cell responses. This study shows that in MC3T3 preosteoblasts focal adhesion kinase (FAK) Y397 phosphorylation, cell spreading, and osteogenic differentiation can be controlled by RGD nanopatterning, with the distribution of islands throughout the hydrogel (i.e. how closely spaced the islands are) being the most significant pattern parameter. More closely spaced islands favor FAK Y397 phosphorylation and cell spreading, while more widely spaced islands favor differentiation. Proliferation, in contrast, is primarily a function of RGD bulk density. Nanopatterning of cell adhesion ligands has tremendous potential as a simple tool to gain significant control over multiple cell behaviors in engineered extracellular matrix (ECM).

Keywords: Hydrogel; Bone tissue engineering; Cell adhesion; Nanopatterns; RGD peptide

The role of polyester interstitium and aldosterone during structural development of renal tubules in serum-free medium by Will W. Minuth; Lucia Denk; Kanghong Hu (pp. 4418-4428).
Little knowledge is available regarding the development of renal stem/progenitor cells into functional parenchyme. To investigate the environmental mechanisms during this maturation process, we elaborated an advanced culture technique to follow renal tubule development. Embryonic stem/progenitor cells derived from neonatal rabbit kidney were placed in a perfusion culture container at the interphase of an artificial polyester interstitium. Tissue culture was carried out in IMDM without serum or protein supplementation and without coating with extracellular matrix proteins. Development of tubules was registered histochemically on cryosections labeled with soybean agglutinin (SBA) and tissue-specific antibodies. The experiments revealed that the development of renal tubules depends exclusively on the administration of aldosterone. The use of 1×10−7m aldosterone for 13 days generated numerous SBA-labeled tubules, while no tubules developed in the absence of the steroid hormone. To obtain further information about the action of the hormone on the cognate receptor, molecular precursors of the aldosterone synthesis pathway were tested. Surprisingly, application of cholesterol, pregnenolone, progesterone, 11-deoxycorticosterone (DOCA) and corticosterone failed to form numerous tubules. Only 11-DOCA and progesterone induced a few tubules, which were barely SBA-labeled. Furthermore, application of aldosterone antagonists such as 1×10−4m spironolactone and 1×10−4m canrenoate completely inhibited the development of tubules. We conclude that specifically aldosterone promotes the development of tubules via the mineralocorticoid receptor whereas its precursors have no effect.

Keywords: Kidney; Tissue engineering; Stem/progenitor cells; Tubules; Artificial interstitium; Aldosterone

Mechanical and structural characterisation of completely degradable polylactic acid/calcium phosphate glass scaffolds by Montse Charles-Harris; Sergio del Valle; Emilie Hentges; Pierre Bleuet; Damien Lacroix; Josep A. Planell (pp. 4429-4438).
This study involves the mechanical and structural characterisation of completely degradable scaffolds for tissue engineering applications. The scaffolds are a composite of polylactic acid (PLA) and a soluble calcium phosphate glass, and are thus completely degradable. A factorial experimental design was applied to optimise scaffold composition prior to simultaneous microtomography and micromechanical testing. Synchrotron X-ray microtomography combined with in situ micromechanical testing was performed to obtain three-dimensional (3D) images of the scaffolds under compression. The 3D reconstruction was converted into a finite element mesh which was validated by simulating a compression test and comparing it with experimental results. The experimental design reveals that larger glass particle and pore sizes reduce the stiffness of the scaffolds, and that the porosity is largely unaffected by changes in pore sizes or glass weight content. The porosity ranges between 93% and 96.5%, and the stiffness ranges between 50 and 200kPa. X-ray projections show a homogeneous distribution of the glass particles within the PLA matrix, and illustrate pore-wall breakage under strain. The 3D reconstructions are used qualitatively to visualise the distribution of the phases of the composite material, and to follow pore deformation under compression. Quantitatively, scaffold porosity, pore interconnectivity and surface/volume ratios have been calculated. Finite element analysis revealed the stress and strain distribution in the scaffold under compression, and could be used in the future to characterise the mechanical properties of the scaffolds.

Keywords: Synchrotron X-ray microtomography; Mechanical test; Biodegradable glass; Scaffold; Finite element analysis

Physical properties of alginate hydrogels and their effects on in vitro follicle development by Erin R. West; Min Xu; Teresa K. Woodruff; Lonnie D. Shea (pp. 4439-4448).
The mechanical properties and density of natural and synthetic extracellular matrices are known to affect cellular processes and regulate tissue formation. In this report, these factors were independently investigated for their role in ovarian follicle development. The matrix composition was controlled through decreasing the solids concentration or the molar mass of the encapsulating biomaterial, alginate. Decreasing matrix stiffness and solids concentration enhanced follicle growth and coordinated differentiation of the follicle cell types, as evidenced by antral cavity formation, theca cell differentiation, oocyte maturation, and relative hormone production levels. While a stiff environment favored high progesterone and androgen secretion, decreasing alginate stiffness resulted in estrogen production which exceeded progesterone and androgen accumulation. These studies reveal, for the first time, a direct link between the biomechanical environment and follicle function, and suggest a novel non-hormonal mechanism regulating follicle development.

Keywords: Alginate; Follicle; Ovary; Tissue engineering; Hydrogel; Encapsulated cells

Silk fibroin matrices for the controlled release of nerve growth factor (NGF) by Lorenz Uebersax; Marta Mattotti; M. Papaloizos Michaël Papaloïzos; Hans P. Merkle; Bruno Gander; Lorenz Meinel (pp. 4449-4460).
Nerve conduits (NC) for peripheral nerve repair should guide the sprouting axons and physically protect the axonal cone from any damage. The NC should also degrade after completion of its function to obviate the need of subsequent explanation and should optionally be suitable for controlled drug release of embedded growth factors to enhance nerve regeneration. Silk fibroin (SF) is a biocompatible and slowly biodegradable biomaterial with excellent mechanical properties that could meet the above stated requirements. SF material (films) supported the adherence and metabolic activity of PC12 cells, and, in combination with nerve growth factor (NGF), supported neurite outgrowth during PC12 cell differentiation. NGF-loaded SF-NC were prepared from aqueous solutions of NGF and SF (20%, w/w), which were air-dried or freeze-dried (freezing at −20 or −196°C) in suitable molds. NGF release from the three differently prepared SF-NC was prolonged over at least 3 weeks, but the total amount released depended on the drying procedure of the NC. The potency of released NGF was retained within all formulations. Control experiments with differently dried NGF-lactose solutions did not evidence marked protein aggregation (SEC, HPLC), loss of ELISA-reactivity or PC12 cell bioactivity. This study encourages the further exploitation of SF-NC for growth factor delivery and evaluation in peripheral nerve repair.

Keywords: Silk fibroin; Nerve conduits; Nerve growth factor; Bioactivity; PC12 cells; SEC

Radiopaque iodinated polymeric nanoparticles for X-ray imaging applications by Anna Galperin; David Margel; Jack Baniel; Gillian Dank; Hagit Biton; Shlomo Margel (pp. 4461-4468).
Iodinated radiopaque polymeric nanoparticles of sizes ranging between 30 and 350nm were formed by emulsion polymerization of the monomer 2-methacryloyloxyethyl(2,3,5-triiodobenzoate) in the presence of sodium dodecyl sulfate as surfactant and potassium persulfate as initiator. The influence of various polymerization parameters, e.g., monomer, initiator and surfactant concentrations on the molecular weight, polymerization yield, size and size distribution of the particles was elucidated. Characterization of these iodinated nanoparticles was accomplished by routine methods such as FTIR,1H NMR, TEM, TGA, DSC, GPC and light scattering. These polymeric nanoparticles are composed of ca. 58% by weight iodine, and are therefore expected to possess significant radiopaque nature. In vitro radiopacity of the iodinated nanoparticles of 30.6±5.0nm diameter, dispersed in water and in the dry state, was demonstrated with a CT scanner. In vivo CT-imaging performed in a dog model by intravenous administration of the uniform 30.6±5.0nm diameter radiopaque nanoparticles dispersed in saline demonstrated significant enhanced visibility of lymph nodes, liver, kidney and spleen. These results indicate that these nanoparticles may be useful as new efficient contrast agents for X-ray imaging applications.

Keywords: Iodine-containing radiopaque nanoparticles; Emulsion polymerization; Radiopacity; X-ray imaging

Adenovirus BMP2-induced osteogenesis in combination with collagen carriers by Zbigniew Gugala; Alan R. Davis; Christine M. Fouletier-Dilling; Francis H. Gannon; Ronald W. Lindsey; Elizabeth A. Olmsted-Davis (pp. 4469-4479).
Adenovirus BMP2 gene therapy has potential of a robust endogenous BMP2 production, while circumventing many of the problems currently associated with recombinant BMP2. The study objective was to determine and compare the ability of adenovirus BMP2 ex vivo gene therapy in combination with three types of collagen carriers to release BMP2 in vitro and to induce heterotopic bone formation in vivo. Human CD45-negative bone marrow cells were ex vivo transduced with a chimeric Ad5F35BMP2. The bioactivity of BMP2 produced by the transduced cells without a carrier, or in combination with three types of collagen carriers (injectable gel, microporous sponge, collagen–mineral composite) was measured and compared to rhBMP2. The heterotopic osteoinductivity assay was performed in immunocompromised NOD/SCID mice. A statistically significant decrease in the amount of rhBMP2 and adenoviral BMP2 released in vitro from the collagen–mineral composite carrier was noted (21% and 12%, respectively), whereas the amounts of rhBMP2 and adenoviral BMP2 released from the gel or sponge carriers were comparable. In vivo, 14 days post-implantation, no bone was formed consistently in groups with the empty Ad5F35HM4 control vector. New bone formation was evident radiographically and histologically in all groups with the Ad5F35BMP2-transduced cells irrespective of the presence or absence of a carrier. The presence of a carrier resulted in osteogenesis limited to the implantation site, and was most pronounced for solid (sponge, composite) carriers. The physical characteristics of the carrier determined the new bone spatial distribution at the site. Solid carriers reduced the clearance of AD5F35-transduced cells by the host immune cells. Adenoviral ex vivo BMP2 gene therapy in combination with collagen carriers with distinct physical characteristics offers the prospects of adjusting this approach to optimally match the specific therapeutic requirements.

Keywords: Osteogenesis; Gene therapy; Bone morphogenetic protein; Adenovirus; Collagen carriers

In vivo analysis of retroviral gene transfer to chondrocytes within collagen scaffolds for the treatment of osteochondral defects by Peter Ueblacker; Bettina Wagner; Stephan Vogt; Gian Salzmann; Gabi Wexel; Kruger Achim Krüger; Christian Plank; Thomas Brill; Karin Specht; Tilla Hennig; Ulrike Schillinger; Andreas B. Imhoff; Vladimir Martinek; Bernd Gansbacher (pp. 4480-4487).
To examine a retroviral gene transfer to chondrocytes in vitro and in vivo in tissue-engineered cell–collagen constructs articular chondrocytes from rabbits and humans were isolated and transduced with VSV.G pseudotyped murine leukemia virus-derived retroviral vectors. Viral supernatants were generated by transient transfection of 293T cells using the pBullet retroviral vector carrying the nlslacZ gene, a Moloney murine leukemia virus gag/pol plasmid and a VSV.G coding plasmid. Transduction efficiency was analyzed by fluorescence-activated-cell-sorter analysis and transduced autologous chondrocytes from rabbits were seeded on collagen-scaffolds and implanted into osteochondral defects in the patellar groove of the rabbit's femur ( n=10). LacZ-expression was analyzed by X-gal staining on total knee explants and histological sections. Retroviral transduction efficiency exceeded 92.3% (SEM±3.5%) in rabbit articular chondrocytes, 74.7% (SEM±1.8%) in human articular chondrocytes and 52.7% (SEM±5.8%) in osteoarthritic human chondrocytes. Reporter gene expression remained high after 15 weeks in 75.7% (SEM±8.2%) of transduced rabbit articular chondrocytes. In vivo, intraarticular β-galactosidase activity could be determined in the majority of implanted chondrocytes in the osteochondral defects after 4 weeks.

Keywords: Chondrocytes; Gene transfer; Retrovirus; Tissue-engineering; Transplantation

The gene transfection efficiency of thermoresponsive N, N, N-trimethyl chitosan chloride-g-poly( N-isopropylacrylamide) copolymer by Zhengwei Mao; Lie Ma; Jiang Yan; Ming Yan; Changyou Gao; Jiacong Shen (pp. 4488-4500).
A thermoresponsive copolymer, trimethyl chitosan-g-poly( N-isopropylacrylamide) (TMC-g-PNIPAAm), was synthesized by coupling PNIPAAm-COOH to TMC. Their molecular structures were characterized by1HNMR. The lower critical solution temperature (LCST) of TMC-g-PNIPAAm in PBS was measured as 32°C by dynamic light scattering (DLS) and UV–vis spectroscopy, regardless of the grafting ratios. Upon mixing with DNA, TMC/DNA particles were formed, whose size and morphology were investigated by DLS and transmission electron microscopy, respectively. The particle size ranged from 200 to 900nm depending on the N/P ratio and was less influenced by the temperature variation. The majority of the particles have spherical morphology. The zeta potentials of these particles were increased along with the N/P ratio. At a given N/P ratio, the zeta potentials were almost constant at 25°C regardless of the existence of serum proteins. However, the values were significantly decreased at 37°C in a solution containing serum protein. The affinity between DNA and TMC was examined by ethidium bromide competitive binding assay. TMC-g-PNIPAAm has stronger ability to combine with DNA at 40°C when the PNIPAAm chain is collapsed. Gel electrophoresis results reveal that the vectors/DNA complexes can be formed regardless of the incubation temperature. HEK293 cell line was chosen as a model to study cellular uptake of the TMC-g-PNIPAAm/DNA particles, gene transfection and cytotoxicity. The grafting of PNIPAAm will not affect cellular uptake of the particles at 37°C. The level of gene transfection could be thermally controlled. By using a temperature variation protocol, i.e. incubation of the cultured cells at 25°C for a while, the gene transfection efficiency was significantly improved. Finally, the optimized gene transfection efficiency achieved by TMC-g-PNIPAAm is comparable to Lipofectamine 2000. No obvious cytotoxicity was detected for the TMC-g-PNIPAAm/DNA particles. These results suggest that TMC-g-PNIPAAm is an effective thermoresponsive gene carrier with minimal cytotoxicity, which has great promise for practical applications.

Keywords: Thermoresponsive; Trimethyl chitosan; Non-viral vector; Gene delivery

Fractal-related assembly of the axial filament in the demosponge Suberites domuncula: Relevance to biomineralization and the formation of biogenic silica by Muller Werner E.G. Müller /; Alexandra Boreiko; Schlobmacher Ute Schloßmacher; Xiaohong Wang; Muhammad Nawaz Tahir; Wolfgang Tremel; David Brandt; Jaap A. Kaandorp; Schroder Heinz C. Schröder (pp. 4501-4511).
The siliceous spicules of sponges (Porifera) show great variations of sizes, shapes and forms; they constitute the chief supporting framework of these animals; these skeletal elements are synthesized enzymatically by silicatein. Each sponge species synthesizes at least two silicateins, which are termed − α and − β. In the present study, using the demosponge Suberites domuncula, we studied if the silicateins of the axial filament contribute to the shape formation of the spicules. For these experiments native silicateins have been isolated by a new Tris/glycerol extraction procedure. Silicateins isolated by this procedure are monomeric (24kDa), but readily form dimers through non-covalent linkages; they show a considerable proteolytic activity that increases during the polymerization phase of the protein. The assembled silicateins (dimers, tetramers as well as hexamers) can be demonstrated in zymograms. The filament/aggregate formation from disassembled silicatein can be visualized by light microscopy and by transmission electron microscopic (TEM) analyses. Since in S. domuncula silicatein- α is four times more abundant in the axial filament than silicatein- β we propose that four silicateins form a platform with serine clusters directed to the center. These serines of the con-axially arranged silicateins interact with silicatein- β. We conclude that initially the silicateins re-assemble chaotically, and in the second phase order themselves to fractal-like structures, which subsequently form the filaments.

Keywords: Sponges; Suberites domuncula; Spicules; Self-assembly; Bio-silica; Silicatein

Influence of environment on the mechanical behaviour of mature human enamel by Li Hong He; Michael V. Swain (pp. 4512-4520).
To investigate the role of different environments in regulating the mechanical behaviour of mature human enamel.Healthy enamel samples were subjected to different environmental treatments such as ethanol dehydration, water re-hydration, desiccation at room temperature, and after heating (burnt) to 300°C. Nanoindentation tests were done on all samples for determination of elastic modulus, hardness and indentation creep behaviour. Scanning electron microscopy (SEM) was used to observe surfaces and indentation impressions of different treated samples.Statistically significant differences of the mechanical properties were found following the various treatments. Burnt sample had the highest elastic modulus and hardness of ∼115 and ∼6GPa, respectively, while the re-hydrated sample showed the lowest values of ∼95 and ∼4GPa. The creep deformation showed the inverse response to the environment-induced elastic modulus results with negligible creep found for the burnt specimens. SEM showed that, although no significant structural changes were found for burnt samples after heating, there was much more cracking about the residual indentation impression.Because of the chemical and thermal stability of hydroxyapatite under the experimental conditions investigated, differences of mechanical behaviour of enamel are rationalized in terms of changes to the matrix proteins and loss of water within enamel. These results indicate that matrix proteins play an important role in regulating the mechanical behaviour of enamel as a biocomposite.

Keywords: Enamel; Nanoindentation; Mechanical properties; Environment

The effect of positively charged plasma polymerization on initial osteoblastic focal adhesion on titanium surfaces by Birgit Finke; Frank Luethen; Karsten Schroeder; Petra D. Mueller; Claudia Bergemann; Marion Frant; Andreas Ohl; Barbara J. Nebe (pp. 4521-4534).
The crucial factor of metal implant ingrowth in the bone is the rapid cellular acceptance. Therefore, the knowledge about additionally used adhesion mechanisms of osteoblasts, like their negatively charged hyaluronan coat, generates new surface functionalization strategies. Here, titanium was coated with a very thin, adherent, cross-linked, pinhole- and additive-free allylamine plasma polymer layer (PPAAm) resistant to hydrolysis and delamination and equipped with a high density of positively charged amino groups. This plasma polymer-functionalization of titanium is advantageous concerning osteoblastic focal adhesion formation as vinculin and paxillin, actin cytoskeleton development and, in consequence in differentiated cell functions, compared to a pure titanium surface—but similar such as the collagen I bonded surface via a polyethylenglycol-diacid (PEG DA)-spacer.

Keywords: Titanium; Surface modification; Plasma polymerization; Hyaluronan; Actin; Adhesion molecules

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