Dense carbon-nanotube coating scaffolds stimulate osteogenic differentiation of mesenchymal stem cells.

Carbon nanotubes (CNTs) have desirable mechanical properties for use as biomaterials in orthopedic and dental area such as bone- and tooth- substitutes. Here, we demonstrate that a glass surface densely coated with single-walled carbon nanotubes (SWNTs) stimulate the osteogenic differentiation of rat bone marrow mesenchymal stem cells (MSCs). MSCs incubated on SWNT- and multi-walled carbon nanotube (MWNT)-coated glass showed high activities of alkaline phosphatase that are markers for early stage osteogenic differentiation. Expression of Bmp2, Runx2, and Alpl of MSCs showed high level in the early stage for MSC incubation on SWNT- and MWNT-coated surfaces, but only the cells on the SWNT-coated glass showed high expression levels of Bglap (Osteocalcin). The cells on the SWNT-coated glass also contained the most calcium, and their calcium deposits had long needle-shaped crystals. SWNT coating at high density could be part of a new scaffold for bone regeneration.

Nanoindentation and finite element modelling of chitosan-alginate multilayer coated hydrogels.

Composite soft materials are used as compounds for determining the effects of mechanical cues on cell behavior and cell encapsulation and for controlling drug release. The appropriate composite soft materials are conventionally prepared by selective deposition of polymers at the surface of an ionic hydrogel. In the present study we address the impact of a mechanically stratified two-layer structure of these materials on their overall mechanical characterization by applying a combination of nanoindentation, confocal microscopy and finite element modelling. We prepare covalent cross-linked hydrogels based on acrylamide (AAM) and including an anionic group, and impregnate them using a multilayer deposition strategy of alternating exposure to cationic chitosan and anionic alginate. The thickness of the chitosan-alginate layer on the hydrogels was determined to be 0.4 ± 0.05 µm for 4 bilayers, and 0.7 ± 0.1 µm for the 8 bilayer deposition procedure employing a fluorescently labelled chitosan and confocal microscopy. The force-indentation data for the AAM gels were highly reproducible, whereas 77% and 50% of the force-indentation data were reproducible following the 4 and 8 bilayer deposition. The main trends in the reproducible force-distance data were found to yield an apparent increased Young's modulus after the deposition. Finite element modelling showed that adaption of a homogeneous Young's modulus for the specimens with deposited layers yields approximately three times too low stiffness compared to the estimate of the mechanical properties of the outer part in the two-layered mechanical model. The thickness of the multilayer region determined by confocal microscopy was used in the model. This study shows that the mechanical layered property needs to be included in the interpretation of the nanoindentation data when there is a significant mechanical contrast.

Delaying cluster growth of ionotropic induced alginate gelation by oligoguluronate.

Alginates form gels in the presence of various divalent ions, such as Ca(2+) that mediate lateral association of chain segments. Various procedures exist that introduce Ca(2+) to yield alginate hydrogels with overall homogeneous or controlled gradients in the concentration profiles. In the present study, the effect of adding oligomers of α-l-guluronic acid (oligoGs) to gelling solutions of alginate was investigated by determination of the cluster growth stimulated by in situ release of Ca(2+). Three different alginate samples varying in fraction of α-l-guluronic acid and molecular weights were employed. The cluster growth was determined for both pure alginates and alginates with two different concentrations of the oligoGs employing dynamic light scattering. The results show that addition of oligoG slows down the cluster growth, the more efficient for the alginates with higher fraction of α-l-guluronic acid, and the higher molecular weight. The efficiency in delaying and slowing the cluster growth induced by added oligoG were discussed in view of the molecular parameters of the alginates. These results show that oligoG can be added to alginate solutions to control the cluster growth and eventually also transition to the gel state. Quantitative relation between the concentration of added oligoG, type and molecular weight of the alginate, and concentration, can be employed as guidelines in tuning alginate cluster growth with specific properties.

The Breast Cancer-Associated Glycoforms of MUC1, MUC1-Tn and sialyl-Tn, Are Expressed in COSMC Wild-Type Cells and Bind the C-Type Lectin MGL.

Aberrant glycosylation occurs in the majority of human cancers and changes in mucin-type O-glycosylation are key events that play a role in the induction of invasion and metastases. These changes generate novel cancer-specific glyco-antigens that can interact with cells of the immune system through carbohydrate binding lectins. Two glyco-epitopes that are found expressed by many carcinomas are Tn (GalNAc-Ser/Thr) and STn (NeuAcα2,6GalNAc-Ser/Thr). These glycans can be carried on many mucin-type glycoproteins including MUC1. We show that the majority of breast cancers carry Tn within the same cell and in close proximity to extended glycan T (Galß1,3GalNAc) the addition of Gal to the GalNAc being catalysed by the T synthase. The presence of active T synthase suggests that loss of the private chaperone for T synthase, COSMC, does not explain the expression of Tn and STn in breast cancer cells. We show that MUC1 carrying both Tn or STn can bind to the C-type lectin MGL and using atomic force microscopy show that they bind to MGL with a similar dead adhesion force. Tumour associated STn is associated with poor prognosis and resistance to chemotherapy in breast carcinomas, inhibition of DC maturation, DC apoptosis and inhibition of NK activity. As engagement of MGL in the absence of TLR triggering may lead to anergy, the binding of MUC1-STn to MGL may be in part responsible for some of the characteristics of STn expressing tumours.

PEGylated chitosan complexes DNA while improving polyplex colloidal stability and gene transfection efficiency.

Chitosan is widely explored as a gene delivery vehicle due to its ability to condense DNA, facilitate transport, and subsequent release allowing gene expression, as well as protecting the DNA. Here, we investigate the enhancement of chitosan-DNA dispersion stability while maintaining transfection efficacy by PEGylation of chitosan. Molecular properties of fully deacetylated chitosans and degree of PEGylation were investigated with respect to compaction of DNA, stability and transfection efficacy. Each of the three chitosan samples with varying chain lengths was PEGylated at three different degrees. The chitosans with degree of PEGylation from 0.6 to 1.9% made polyplexes with DNA. PBS induced colloidal aggregation of polyplexes with initial radius of about 100 nm observed for nonPEGylated chitosans was suppressed for 1.9% PEGylated chitosans. The observed increase in transfection efficacy coinciding with increased polyplex colloidal stability suggests that aggregation of gene-delivery packages may reduce the transfection efficacy.

Isothermal titration calorimetry study of the polyelectrolyte complexation of xanthan and chitosan samples of different degree of polymerization.

Mixing oppositely charged polyelectrolytes in aqueous solutions leads to the spontaneous formation of polyelectrolyte complexes. Here, we characterize the interaction between xanthan of two different chain lengths, a tri-glucosamine and a chitosan polymer by isothermal titration calorimetry (ITC). Analysis of the experimental thermodynamic data assuming a single set of identical sites indicated both enthalpic and entropic contributions to the overall interaction in the interaction between xanthan and tri-glucosamine. The relative contribution of entropy compared to enthalpy was found to be largest for the shortest chain length of xanthan. Using a chitosan polymer instead of tri-glucosamine gave rise to two different stages in the interaction process. A model where the first stage of the ITC curve represent an initial polyelectrolyte complexation stage followed by aggregation on further titration of chitosan to the xanthan is suggested. Ultrastructure images by applying atomic force microscopy at some selected extents of titration are consistent with the two-stage interpretation of the thermodynamic data.

Dehydration stability of amyloid fibrils studied by AFM.

Atomic force microscopy was used to investigate the stability of dehydrated amyloid fibrils formed by human islet polypeptide (IAPP) and Abeta(1-42) peptides. IAPP amyloid fibrils were imaged in liquid (hydrated state) and in air (dehydrated). In addition, fibrils dried on the mica surface were rehydrated and re-examined both in liquid and in air (after consecutive redrying). As reported previously, the initial drying process does not result in any major change in the amyloid appearance and the dimensions of the fibrils are preserved. However, when once-dried samples are rehydrated, fibril stability is lost. The fibrils disintegrate into small particles that are attached to the mica surface. This process is further confirmed by studies of the rehydrated samples after drying, on which the morphology of the fibrils is clearly changed. Similar behavior is observed for Abeta(1-42) amyloid fibrils, which are apparently stable on first drying, but disintegrate on rehydration. The observed change indicates that dehydration is causing a change in the internal structure of the amyloid fibrils. This has important implications for studies of amyloid fibrils by other techniques. Due to the potential influence of hydration and sample history on amyloid structure, preparation and study of amyloid samples with controlled humidity requires more consideration.

Characterizing DNA condensation by structurally different chitosans of variable gene transfer efficacy.

Chitosan can be used as a nonviral gene delivery vector for which DNA condensation and transfection efficacy strongly depend on structural parameters. In this study, we characterized the condensation of DNA by three molecularly tailored chitosans, including linear, trisaccharide substituted-, and self-branched trisaccharide substituted chitosan oligomers. No significant differences could be detected in the hydrodynamic diameters formed by the various chitosans as analyzed by dynamic light scattering. However, atomic force microscopy revealed that self-branched chitosan formed complexes with a higher ratio of globules to rods, and the heights of both globules and rods were larger than for complexes formed by the other chitosans. Using an amino/phosphate ratio of 10, fluorescence correlation spectroscopy measurements showed that self-branched chitosan exhibited a lower fraction (30%) of bound chitosan than the other chitosans. YOYO-1 was a superior fluorescent DNA-label compared to Cy5 and PicoGreen, since labeling with YOYO-1 had least effect on the size and structure of the complexes.

The influence of charge density of chitosan in the compaction of the polyanions DNA and xanthan.

In this study the relative importance of valence and charge density of the polycation chitosan on the compaction process of DNA and xanthan is investigated. Chitosans with approximately equal valence but differing in their charge density were employed to form polyelectrolyte complexes with the two polyanions. The resulting structures (toroids, rods, and globules) have been visualized by AFM. For DNA-chitosan the complexation process was additionally studied by utilizing the fluorescent probe ethidium bromide. The results show that not only the total charge per chitosan molecule (valence), but also the charge density is important in determining the association with polyanions such as DNA and xanthan. Furthermore, it is demonstrated that the pH at which the complexation takes place is an important parameter in the complexation process, influencing the structures formed.

DNA-polycation complexation and polyplex stability in the presence of competing polyanions.

Polyelectrolyte complex (polyplex) formation was studied by employing tapping mode atomic force microscopy (AFM) and an ethidium bromide fluorescence assay. The polycations chitosan and poly-L-lysine were used to compact DNA and the stability of the polyplexes was evaluated upon exposure to competing polyanions (alginate and xanthan). Furthermore, the relative preference of these polycations for DNA and the competing polyanion was investigated. The results showed that neither poly-L-lysine nor chitosan displayed any selectivity in binding to DNA relative to the competing polyanions, demonstrating the importance of electrostatics in the binding of a polycation to a polyanion. However, the ability of the polyanions to destabilize the DNA-polycation complexes depended on both the polyanion and the polycation employed, indicating that polymer-specific properties are also important for the complexation behavior and polyplex stability. Destabilization experiments further showed that annealing yielded complexes that were less prone to disruption upon subsequent exposure to alginate. Annealing experiments of plasmid DNA-chitosan complexes showed an increased fraction of rods following temperature treatment, indicating that the rods most likely are the more stable morphology for this system.

Metastable and stable states of xanthan polyelectrolyte complexes studied by atomic force microscopy.

The compaction of the semiflexible polysaccharide xanthan with selected multi- and polyvalent cations was studied. Polyelectrolyte complexes prepared at concentrations of 1-2 microg/ml were observed by tapping mode atomic force microscopy. High-molecular-weight xanthan compacted with chitosan yields a blend of mainly toroidal and metastable structures and a small fraction of rod-like species. Polyelectrolyte complexes of xanthan with polyethylenimine and trivalent chromium yielded similar structures or alternatively less well packed species. Racquet-type morphologies were identified as kinetically trapped states occurring on the folding path toward the energetically stable state of the toroids. Thermal annealing yielded a shift of the distribution of xanthan-chitosan morphologies toward this stable state. Ensembles of toroidal and rod-like morphologies of the xanthan-chitosan structures, collected using an asphericity index, were analyzed. The mean height of the toroids increased upon heating, with a selective increase in the height range above 2 nm. It is suggested that the observed metastable structures are formed from the high-molecular-weight fraction of xanthan and that these are driven toward the toroidal state, being a low-energy state, following annealing. Considered a model system for condensation of semiflexible polymers, the compaction of xanthan by chitosan captures the system at various stages in the folding toward a low-energy state and thus allows experimental analyses of these intermediates and their evolution.

Characterisation of bacterial polysaccharides: steps towards single-molecular studies.

Techniques used in studies of polysaccharides, including chemical composition, linkage pattern, and higher order structures are in constant development. They provide information necessary for understanding of the polysaccharide properties and functions. Here, recent advancements in studies of the polysaccharides at the single-molecule level are highlighted. Over the last few years, single-molecule techniques such as force spectroscopy have improved in sensitivity and can today be used to detect forces in the pN range. In addition, these techniques can be used to investigate properties of single molecules close to physiological conditions. The challenges in the interpretation of the observations are aided by control experiments using well-characterised polysaccharides and by data provided by complementary methods. This field is expected to have increasing impact on the further advancement of the molecular understanding of the role of polysaccharides in various biological processes such as recognition and cell adhesion.

Cardiac and pulmonary doses and complication probabilities in standard and conformal tangential irradiation in conservative management of breast cancer.

BACKGROUND AND PURPOSE: The clinical benefit of irradiating the intact breast after lumpectomy must be weighted against the risk of severe toxicity. We present a study on cardiac and pulmonary dose-volume data and the related complication probabilities of tangential breast irradiation having the following objectives: (1) to quantify the sparing of the organs at risk (ORs), the heart and the lung, achieved by three-dimensional (3-D) conformal tangential irradiation (CTI) as compared to standard tangential irradiation (STI); (2) to elucidate the uncertainty in radiation tolerance data; and (3) to analyse the relation between the amount of OR irradiated and the resulting morbidity risk. MATERIAL AND METHODS: Computed tomography (CT)-based 3-D treatment plans of 26 patients prescribed to CTI of the intact breast were applied. Contour-based STI has been our routine treatment, and was reconstructed for all patients. Dose-volume data and normal tissue complication probability (NTCP) predictions from the probit and relative seriality models with several cardiac and pulmonary tolerance parameterizations were analysed and compared. RESULTS AND CONCLUSIONS: A significant amount of normal tissues can be spared from radiation by using CT-based CTI, resulting in a 50% reduction of the average excess cardiac mortality risk in the left-sided cases. The risks for pericarditis and pneumonitis were too low to reveal any clinically significant difference between the treatments. For the STI set-up, a regression analysis showed that the excess cardiac mortality risk increased when larger parts of the heart were inside the fields. However, the different excess cardiac mortality and pneumonitis tolerance parameters resulted in statistically significant different NTCPs, which precluded the ability to accurately predict absolute NTCPs after tangential breast irradiation. Despite this uncertainty the different series of cardiac and pulmonary risk predictions were in relatively good agreement when small volumes of the ORs were irradiated. From the present data and without consideration of patient or organ motion, it therefore appears that tangential breast irradiation with less than 1 cm of the heart and 2-2.5 cm of the lung included inside the treatment fields will cause at most 1 per thousand risk for cardiac mortality and pulmonary morbidity. CT-based CTI should be considered, in particular for the left-sided cases, if these requirements cannot be met.