New self-assembling multifunctional templates for the biofabrication and controlled self-release of cultured tissue

The need to source live human tissues for research and clinical applications has been a major driving force for the development of new biomaterials. Ideally, these should elicit the formation of scaffold-free tissues with native-like structure and composition. In this study, we describe a biologically interactive coating that combines the fabrication and subsequent self-release of live purposeful tissues using template–cell–environment feedback. This smart coating was formed from a self-assembling peptide amphiphile comprising a proteasecleavable sequence contiguous with a cell attachment and signaling motif. This multifunctional material was subsequently used not only to instruct human corneal or skin fibroblasts to adhere and deposit discreet multiple layers of native extracellular matrix but also to govern their own self-directed release from the template solely through the action of endogenous metalloproteases. Tissues recovered through this physiologically relevant process were carrier-free and structurally and phenotypically equivalent to their natural counterparts. This technology contributes to a new paradigm in regenerative medicine, whereby materials are able to actively direct and respond to cell behavior. The novel application of such materials as a coating capable of directing the formation and detachment of complex tissues solely under physiological conditions can have broad use for fundamental research and in future cell and tissue therapies.

Stability of probiotic Lactobacillus plantarum in dry microcapsules under accelerated storage conditions

This study investigated the stability of freeze dried and fluid bed dried alginate microcapsules coated with chitosan containing model probiotic bacteria, Lactobacillus plantarum, during storage for up to 45 days at different water activities (0.11, 0.23, 0.40 and 0.70) and temperatures (4, 30 and 37 °C). The loss in cell viability was around 0.8 log in the case of fluid bed drying and around 1.3 in the case of freeze drying, with the former method resulting in dried capsules of smaller size (~ 1 mm vs 1.3 mm), more irregular shape, and with a rougher surface. In both cases, the water activity and water content were less than 0.25 and 10% w/w, respectively, which favours high storage stability. The storage stability studies demonstrated that as the water activity and temperature decreased the survival of the dried encapsulated cells increased. Considerably better survival was observed for fluid bed dried encapsulated cells compared to freeze dried encapsulated cells and freeze dried free cells with 10% sucrose (control), and in some cases, e.g. at 4 and 30 °C at water activities of 0.11, 0.23 and 0.40, there was more than 1 log difference after 45 days, with concentrations higher than 108 CFU/g after 45 days of storage. The results indicate that fluid bed drying is an effective and efficient manufacturing method to produce probiotic containing capsules with enhanced storage stability.

Human neural stem cell-derived cultures in three- dimensional substrates form spontaneously functional neuronal networks

Differentiated human neural stem cells were cultured in an inert three-dimensional (3D) scaffold and, unlike two-dimensional (2D) but otherwise comparable monolayer cultures, formed spontaneously active, functional neuronal networks that responded reproducibly and predictably to conventional pharmacological treatments to reveal functional, glutamatergic synapses. Immunocytochemical and electron microscopy analysis revealed a neuronal and glial population, where markers of neuronal maturity were observed in the former. Oligonucleotide microarray analysis revealed substantial differences in gene expression conferred by culturing in a 3D vs a 2D environment. Notable and numerous differences were seen in genes coding for neuronal function, the extracellular matrix and cytoskeleton. In addition to producing functional networks, differentiated human neural stem cells grown in inert scaffolds offer several significant advantages over conventional 2D monolayers. These advantages include cost savings and improved physiological relevance, which make them better suited for use in the pharmacological and toxicological assays required for development of stem cell-based treatments and the reduction of animal use in medical research.

Mesenchymal stem cells and inorganic bovine bone mineral in sinus augmentation: comparison with augmentation by autologous bone in adult sheep

Our aim was to compare the osteogenic potential of mononuclear cells harvested from the iliac crest combined with bovine bone mineral (BBM) (experimental group) with that of autogenous cancellous bone alone (control group). We studied bilateral augmentations of the sinus floor in 6 adult sheep. BBM and mononuclear cells (MNC) were mixed and placed into one side and autogenous bone in the other side. Animals were killed after 8 and 16 weeks. Sites of augmentation were analysed radiographically and histologically. The mean (SD) augmentation volume was 3.0 (1.0) cm(3) and 2.7 (0.3) cm(3) after 8 and 16 weeks in the test group, and 2.8 (0.3) cm(3) (8 weeks) and 2.8 (1.2) cm(3) (16 weeks) in the control group, respectively. After 8 weeks, histomorphometric analysis showed 24 (3)% BBM, and 19 (11)% of newly formed bone in the test group. The control group had 20 (13%) of newly formed bone. Specimens after 16 weeks showed 29 (12%) of newly formed bone and 19 (3%) BBM in the test group. The amount of newly formed bone in the control group was 16 (6%). The results show that mononuclear cells, including mesenchymal stem cells, in combination with BBM as the biomaterial, have the potential to form bone. (C) 2009 The British Association of Oral and Maxillofacial Surgeons. Published by Elsevier Ltd. All rights reserved.

Bone Formation Following Implantation of Titanium Sponge Rods into Humeral Osteotomies in Dogs: A Histological and Histometrical Study

Background: Titanium (Ti) is widely proven to enhance bone contact and growth on its surface. It is expected that bone defects could benefit from Ti to promote healing and to increase strength of the implanted area. Purpose: The present study aimed at comparing the potential of porous Ti sponge rods with synthetic hydroxyapatite (HA) for the healing of bone defects in a canine model. Material and Methods: Six mongrel dogs were submitted to three trephined osteotomies of 6.0 x 4.0 mm in one humerus and after 2 months another three osteotomies were performed in the contralateral humerus. A total of 36 defects were randomly filled either with Ti foam, particulate HA, or coagulum (control). The six animals were killed 4 months after the first surgery for histological and histometrical analysis. Results: The Ti-foam surface was frequently found in intimate contact with new bone especially at the defect walls. Control sites showed higher amounts of newly formed bone at 2 months - Ti (p = 0.000) and HA (p = 0.009) - and 4 months when compared with Ti (p = 0.001). Differently from HA, the Ti foam was densely distributed across the defect area which rendered less space for bone growth in the latter`s sites. The use of Ti foams or HA resulted in similar amounts of bone formation in both time intervals. Nevertheless, the presence of a Ti-foam rod preserved defect`s marginal bone height as compared with control groups. Also, the Ti-foam group showed a more mature bone pattern at 4 months than HA sites. Conclusion: The Ti foam exhibited good biocompatibility, and its application resulted in improved maintenance of bone height compared with control sites. The Ti foam in a rod design exhibited bone ingrowth properties suitable for further exploration in other experimental situations.

Strategies to Optimize Biosensors Based on Impedance Spectroscopy to Detect Phytic Acid Using Layer-by-Layer Films

Impedance spectroscopy has been proven a powerful tool for reaching high sensitivity in sensor arrays made with nanostructured films in the so-called electronic tongue systems, whose distinguishing ability may be enhanced with sensing units capable of molecular recognition. In this study we show that for optimized sensors and bio-sensors the dielectric relaxation processes involved in impedance measurements should also be considered, in addition to an adequate choice of sensing materials. We used sensing units made from layer-by-layer (LbL) films with alternating layers of the polyeletrolytes, poly(allylamine) hydrochloride (PAH) and poly(vinyl sulfonate) (PVS), or LbL films of PAH alternated with layers of the enzyme phytase, all adsorbed on gold interdigitate electrodes. Surprisingly, the detection of phytic acid was as effective in the PVS/PAH sensing system as with the PAH/phytase system, in spite of the specific interactions of the latter. This was attributed to the dependence of the relaxation processes on nonspecific interactions such as electrostatic cross-linking and possibly on the distinct film architecture as the phytase layers were found to grow as columns on the LbL film, in contrast to the molecularly thin PAH/PVS films. Using projection techniques, we were able to detect phytic acid at the micromolar level with either of the sensing units in a data analysis procedure that allows for further optimization.

Immobilization of Alcohol Dehydrogenase in Phospholipid Langmuir-Blodgett Films To Detect Ethanol

Enzyme immobilization in nanostructured films may be useful for a number of biomimetic systems, particularly if suitable matrixes are identified. Here we show that alcohol dehydrogenase (ADH) has high affinity toward a negatively charged phospholipid, dimyristoylphosphatidic acid (DMPA), which forms a Langmuir monolayer at an air-water interface. Incorporation of ADH into the DMPA monolayer was monitored with Surface pressure measurements; and polarization-modulation infrared reflection absorption spectroscopy, with the alpha-helices from ADH being mainly oriented parallel to the water surface. ADH remained at the interface even at high surface pressures, thus allowing deposition of Langmuir-Blodgett (LB) films from the DMPA-ADH film. Indeed, interaction with DMPA enhances the transfer of ADH, where the mass transferred onto a solid support increased from 134 ng for ADH on a Gibbs monolayer to 178 ng for an LB film with DMPA. With fluorescence spectroscopy it was possible to confirm that the ADH structure was preserved even after one month of the LB deposition. ADH-containing films deposited onto gold-interdigitated electrodes were employed in a sensor array capable of detecting ethanol at concentrations down to 10 ppb (in volume), using impedance spectroscopy as the method of detection.

Interaction of polysaccharide-protein complex from Agaricus blazei with Langmuir and Langmuir-Blodgett films of phospholipids

The use of natural substances in health applications may be hampered by the difficulties in establishing the mechanisms of action, especially at molecular-level. The protein-polysaccharide complex extracted from the mushroom Agaricus blazei Murill, referred to as CAb, has been considered for treating various diseases with probable interaction with cell membranes. In this study, we investigate the interaction between CAb and a cell membrane model represented by a Langmuir monolayer of dimyristoyl phosphatidic acid (DMPA). CAb affects the structural properties of DMPA monolayers causing expansion and increasing compressibility. In addition, interaction with DMPA polar heads led to neutralization of the electrical double layer, yielding a zero surface potential at large areas per molecule. CAb remained at the interface even at high surface pressures, which allowed transfer of Langmuir-Blodgett (LB) films onto solid supports with the CAb-DMPA mixture. The mass transferred, according to quartz crystal microbalance (QCM) measurements, increased linearly with the number of deposited layers. With UV-vis absorption, fluorescence and FTIR spectroscopies, we confirmed that the LB films contain polysaccharides, proteins and DMPA. Therefore, the CAb biological action must be attributed not only to polysaccharides but also to proteins in the complex. (C) 2008 Elsevier Inc. All rights reserved.

Mixing alternating copolymers containing fluorenyl groups with phospholipids to obtain Langmuir and Langmuir-Blodgett films

The control of molecular architectures may be essential to optimize materials properties for producing luminescent devices from polymers, especially in the blue region of the spectrum. In this Article, we report on the fabrication of Langmuir-Blodgett (LB) films of polyfluorene copolymers mixed with the phospholipid dimyristoyl phosphatidic acid (DMPA). The copolymers poly(9.9-dioetylfluorene)-co-phenylene (copolymer I) and poly(9,9-dioctylfluorene)-co-quaterphenylene) (copolymer 2) were synthesized via Suzuki reaction. Copolymer I could not form a monolayer on its own, but it yielded stable films when mixed with DMPA. In contrast, Langmuir monolayers could be formed from either the neat copolymer 2 or when mixed with DMPA. The surface pressure and surface potential measurements, in addition to Brewster angle microscopy, indicated that DMPA provided a suitable matrix for copolymer I to form a stable Langmuir film, amenable to transfer as LB films, while enhancing the ability of copolymer 2 to form LB films with enhanced emission, as indicated by fluorescence spectroscopy. Because a high emission was obtained with the mixed LB films and since the molecular-level interactions between the film components can be tuned by changing the experimental conditions to allow For further optimization, one may envisage applications of these films in optical devices such as organic light-emitting diodes (OLEDs).

Electrospinning of Hyperbranched Poly-L-Lysine/Polyaniline Nanofibers for Application in Cardiac Tissue Engineering

Electrospun polyaniline nanofibers are one of the most promising materials for cardiac tissue engineering due to their tunable electroactive properties. Moreover, the biocompatibility of polyaniline nanofibes can be improved by grafting of adhesive peptides during the synthesis. In this paper, we describe the biocompatible properties and cardiomyocytes proliferation on polyaniline electrospun nanofibers modified by hyperbranched poly-L-lysine dendrimers (HPLys). The microstructure characterization of the HPLys/polyaniline nanofibers was carried out by scanning electron microscopy (SEM). It was observed that the application of electrical current stimulates the differentiation of cardiac cells cultured on the nanofiber scaffolds. Both electroactivity and biocompatibility of the HPLys based nanofibers suggest the use this material for culture of cardiac cells and opens the possibility of using this material as a biocompatible electroactive 3-D matrix in cardiac tissue engineering.

Structural and thermodynamic analysis of thrombin:suramin interaction in solution and crystal phases

Suramin is a hexasulfonated naphthylurea which has been recently characterized as a non-competitive inhibitor of human alpha-thrombin activity over fibrinogen, although its binding site and mode of interaction with the enzyme remain elusive. Here, we determined two X-ray structure of the thrombin: suramin complex, refined at 2.4 angstrom resolution. While a single thrombin: suramin complex was found in the asymmetric unit cell of the crystal, some of the crystallographic contacts with symmetrically related molecules are mediated by both the enzyme and the ligand. Molecular dynamics simulations with the 1:1 complex demonstrate a large rearrangement of suramin in the complex, but with the protein scaffold and the more extensive protein-ligand regions keep unchanged. Small-angle X-ray scattering measurements at high micromolar concentration demonstrate a suramin-induced dimerization of the enzyme. These data indicating a dissimilar binding mode in the monomeric and oligomeric states, with a monomeric, 1:1 complex to be more likely to exist at the thrombin physiological, nanomolar concentration range. Collectively, close understanding on the structural basis for interaction is given which might establish a basis for design of suramin analogues targeting thrombin. Crown Copyright (C) 2009 Published by Elsevier B.V. All rights reserved.

The three-dimensional structure of bothropasin, the main hemorrhagic factor from Bothrops jararaca venom: Insights for a new classification of snake venom metalloprotease subgroups

Bothropasin is a 48 kDa hemorrhagic PIII snake venom metalloprotease (SVMP) isolated from Bothrops jararaca, containing disintegrin/cysteine-rich adhesive domains. Here we present the crystal structure of bothropasin complexed with the inhibitor POL647. The catalytic domain consists of a scaffold of two subdomains organized similarly to those described for other SVMPs, including the zinc and calcium-binding sites. The free cysteine residue Cys(189) is located within a hydrophobic core and it is not available for disulfide bonding or other interactions. There is no identifiable secondary structure for the disintegrin domain, but instead it is composed mostly of loops stabilized by seven disulfide bonds and by two calcium ions. The ECD region is in a loop and is structurally related to the RGD region of RGD disintegrins, which are derived from I`ll SVMPs. The ECD motif is stabilized by the Cys(117)_Cys(310) disulfide bond (between the disintegrin and cysteine-rich domains) and by one calcium ion. The side chain of Glu(276) of the ECD motif is exposed to solvent and free to make interactions. In bothropasin, the HVR (hyper-variable region) described for other Pill SVMPs in the cysteine-rich domain, presents a well-conserved sequence with respect to several other Pill members from different species. We propose that this subset be referred to as PIII-HCR (highly conserved region) SVMPs. The differences in the disintegrin-like, cysteine-rich or disintegrin-like cysteine-rich domains may be involved in selecting target binding, which in turn could generate substrate diversity or specificity for the catalytic domain. (C) 2008 Elsevier Ltd. All rights reserved.

Xyloglucan nano-aggregates: Physico-chemical characterisation in buffer solution and potential application as a carrier for camptothecin, an anti-cancer drug

In this work, native xyloglucan was extracted from Tamarindus indica seeds (XGT), and its properties in phosphate buffer solution (PBS) were evaluated in comparison with a commercial tamarind kernel powder (TKP). The physico-chemical characteristics of the polysaccharides such as molar mass, critical concentration and intrinsic viscosity were determined. Furthermore, using spectroscopic and microscopy techniques, it was observed that the XGs tested can be considered macromolecules able to aggregate as nano-entities of 60-140 nm. The XGT tended to an ordered and compact spherical conformation determined by the Huggins constant, circular dichroism, atomic force microscopy and transmission electron microscopy. After the determination of the properties in PBS the XGs, at concentrations of 25% above their critical aggregation concentration, were used to encapsulate camptothecin, an anti-cancer drug. The XGT sample showed an encapsulation efficiency of 42% and first-order drug delivery kinetics. These results demonstrated the importance of knowledge of the physico-chemical properties of polysaccharides, for example, to better conduct their biotechnological applications as drug carriers. (C) 2010 Elsevier Ltd. All rights reserved.

Discovery of New Inhibitors of Schistosoma mansoni PNP by Pharmacophore-Based Virtual Screening

Schistosomiasis is considered the second most important tropical parasitic disease, with severe socioeconomic consequences for millions of people worldwide. Schistosoma monsoni, one of the causative agents of human schistosomiasis, is unable to synthesize purine nucleotides de novo, which makes the enzymes of the purine salvage pathway important targets for antischistosomal drug development. In the present work, we describe the development of a pharmacophore model for ligands of S. mansoni purine nucleoside phosphorylase (SmPNP) as well as a pharmacophore-based virtual screening approach, which resulted in the identification of three thioxothiazolidinones (1-3) with substantial in vitro inhibitory activity against SmPNP. Synthesis, biochemical evaluation, and structure activity relationship investigations led to the successful development of a small set of thioxothiazolidinone derivatives harboring a novel chemical scaffold as new competitive inhibitors of SmPNP at the low-micromolar range. Seven compounds were identified with IC(50) values below 100 mu M. The most potent inhibitors 7, 10, and 17 with 1050 of 2, 18, and 38 mu M, respectively, could represent new potential lead compounds for further development of the therapy of schistosomiasis.

XRCC1 haploinsufficiency in mice has little effect on aging, but adversely modifies exposure-dependent susceptibility

Oxidative DNA damage plays a role in disease development and the aging process. A prominent participant in orchestrating the repair of oxidative DNA damage, particularly single-strand breaks, is the scaffold protein XRCC1. A series of chronological and biological aging parameters in XRCC1 heterozygous (HZ) mice were examined. HZ and wild-type (WT) C57BL/6 mice exhibit a similar median lifespan of similar to 26 months and a nearly identical maximal life expectancy of similar to 37 months. However, a number of HZ animals (7 of 92) showed a propensity for abdominal organ rupture, which may stem from developmental abnormalities given the prominent role of XRCC1 in endoderm and mesoderm formation. For other end-points evaluated-weight, fat composition, blood chemistries, condition of major organs, tissues and relevant cell types, behavior, brain volume and function, and chromosome and telomere integrity-HZ mice exhibited by-and-large a normal phenotype. Treatment of animals with the alkylating agent azoxymethane resulted in both liver toxicity and an increased incidence of precancerous lesions in the colon of HZ mice. Our study indicates that XRCC1 haploinsufficiency in mammals has little effect on chronological longevity and many key biological markers of aging in the absence of environmental challenges, but may adversely affect normal animal development or increase disease susceptibility to a relevant genotoxic exposure.