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Biphasic calcium phosphate (BCP) materials are widely employed as bone substitute materials due to their resorption/degradation properties. Inflammation after implantation of such materials represents a prerequisite for bone tissue repair and regeneration but can be also problematic if it is not only transient and if it is followed by fibrosis and scarring. Here, we modified BCP covalently with hyaluronan (HA) and heparin (Hep), glycosaminoglycans that possess anti-inflammatory properties. Beside the characterization of particle surface properties, the focus was on in vivo tissue response after subcutaneous implantation in mice. Histological analysis revealed a decrease in signs of inflammatory response to BCP when modified with either HA or Hep. Reduced vascularization after 30 days was noticed when BCP was modified with either HA or Hep with greater cellularity in all examined time points. Compared to plain BCP, expression of endothelial-related genes Flt1 and Vcam1 was higher in BCP-HA and BCP-Hep group at day 30. Expression of osteogenesis-related genes Sp7 and Bglap after 30 days was the highest in BCP group, followed by BCP-Hep, while the lowest expression was in BCP-HA group which correlates with collagen amount. Hence, coating of BCP particles with HA seems to suppress inflammatory response together with formation of new bone-like tissue, while the presence of Hep delays the onset of inflammatory response but permits osteogenesis in this subcutaneous bone-forming model. Transferring the results of this study to other coated materials intended for biomedical application may also pave the way to reduction of inflammation after their implantation.

Polyelectrolyte multilayers (PEMs) consisting of the polysaccharides hyaluronic acid (HA) as the polyanion and chitosan (Chi) as the polycation were prepared with layer-by-layer technique (LbL). The [Chi/HA]5 multilayers were exposed to solutions of metal ions (Ca2+, Co2+, Cu2+ and Fe3+). Binding of metal ions to [Chi/HA]5 multilayers by the formation of complexes with functional groups of polysaccharides modulates their physical properties and the bioactivity of PEMs with regard to the adhesion and function of multipotent murine C3H10T1/2 embryonic fibroblasts. Characterization of multilayer formation and surface properties using different analytical methods demonstrates changes in the wetting, surface potential and mechanical properties of multilayers depending on the concentration and type of metal ion. Most interestingly, it is observed that Fe3+ metal ions greatly promote adhesion and spreading of C3H10T1/2 cells on the low adhesive [Chi/HA]5 PEM system. The application of intermediate concentrations of Cu2+, Ca2+ and Co2+ as well as low concentrations of Fe3+ to PEMs also results in increased cell spreading. Moreover, it can be shown that complex formation of PEMs with Cu2+ and Fe3+ ions leads to increased metabolic activity in cells after 24 h and induces cell differentiation towards adipocytes in the absence of any additional adipogenic media supplements. Overall, complex formation of [Chi/HA]5 PEM with metal ions like Cu2+ and Fe3+ represents an interesting and cheap alternative to the use of growth factors for making cell-adhesive coatings and guiding stem cell differentiation on implants and scaffolds to regenerate connective-type of tissues.

Laser interference lithography (LIL) and the layer-by-layer (LbL) technique are combined here for the first time to design a system with variable nanotopographies and surface viscoelasticity to regulate cell behavior. LIL is used to generate hexagonally arranged nanostructures of gold with different periodicity. In contrast, LBL is used to assemble a multilayer system of poly-L-lysine and hyaluronic acid on top of the nanostructures. Moreover, the viscoelastic properties of that system are controlled by chemical cross-linking. We show that the topography designed with LIL is still present after multilayer deposition and that the formation of the multilayer system renders the surfaces hydrophilic, which is opposite to the hydrophobic nature of pristine nanostructures. The heterogenic system is applied to study the effect on adhesion and differentiation of human adipose-derived stem cells (hADSC). We show that hADSC spreading is increasing with cross-linking degree on flat multilayers, while it is decreasing on nanostructures modified with multilayers. In addition, early effects on signal transduction processes are seen. Finally, hADSC differentiation into chondrogenic and osteogenic lineages is superior to adipogenic lineages on nanostructures modified with multilayers. Hence, the presented system offers great potential to guide stem cell differentiation on surfaces of implants and tissue engineering scaffolds.

Elastin is an essential structural protein in the extracellular matrix of vertebrates. It is the core component of elastic fibers, which enable connective tissues such as those of the skin, lungs or blood vessels to stretch and recoil. This function is provided by elastin's exceptional properties, which mainly derive from a unique covalent cross-linking between hydrophilic lysine-rich motifs of units of the monomeric precursor tropoelastin. To date, elastin's cross-linking is poorly investigated. Here, we purified elastin from human tissue and cleaved it into soluble peptides using proteases with different specificities. We then analyzed elastin's molecular structure by identifying unmodified residues, post-translational modifications and cross-linked peptides by high-resolution mass spectrometry and amino acid analysis. The data revealed the presence of multiple isoforms in parallel and a complex and heterogeneous molecular interconnection. We discovered that the same lysine residues in different monomers were simultaneously involved in various cross-link types or remained unmodified. Furthermore, both types of cross-linking domains, Lys-Pro and Lys-Ala domains, participate not only in bifunctional inter- but also in intra-domain cross-links. We elucidated the sequences of several desmosine-containing peptides and the contribution of distinct domains such as 6, 14 and 25. In contrast to earlier assumptions proposing that desmosine cross-links are formed solely between two domains, we elucidated the structure of a peptide that proves a desmosine formation with participation of three Lys-Ala domains. In summary, these results provide new and detailed insights into the cross-linking process, which takes place within and between human tropoelastin units in a stochastic manner.

Background: Cells possess a specialized machinery through which they can sense physical as well as chemical alterations in their surrounding microenvironment that affect their cellular behavior. Aim: In this study, we aim to establish a polyelectrolyte multilayer system of 24 layers of poly-l-lysine and hyaluronic acid to control stem cell response after chemical cross-linking. Methods and results: The multilayer build-up process is monitored using different methods, which show that the studied polyelectrolyte multilayer system grows exponentially following the islands and islets theory. Successful chemical cross-linking is monitored by an increased zeta potential toward negative magnitude and an extraordinary growth in thickness. Human adipose-derived stem cells are used here and a relationship between cross-linking degree and cell spreading is shown as cells seeded on higher cross-linked polyelectrolyte multilayer show enhanced spreading. Furthermore, cells that fail to establish focal adhesions on native and low cross-linked polyelectrolyte multilayer films do not proliferate to a high extent in comparison to cells seeded on highly cross-linked polyelectrolyte multilayer, which also show an increased metabolic activity. Moreover, this study shows the relation between cross-linking degree and human adipose-derived stem cell lineage commitment. Histological staining reveals that highly cross-linked polyelectrolyte multilayers support osteogenic differentiation, whereas less cross-linked and native polyelectrolyte multilayers support adipogenic differentiation in the absence of any specific inducers. Conclusion: Owing to the precise control of polyelectrolyte multilayer properties such as potential, wettability, and viscoelasticity, the system presented here offers great potential for guided stem cell differentiation in regenerative medicine, especially in combination with materials exhibiting a defined surface topography.

Glycosaminoglycans (GAGs) comprise the closest cellular environment: they are building elements of the ECM and can be also found on cells surface. Their biological activity depends on several parameters among which the negative charge is of prime importance[1]. This charge is generally associated with the presence of sulfate groups (-OSO3H). Sulfation is a dynamic modification: it can occur at various positions within the glycan and different sulfation patterns have been identified for the same organs and cells during their development. However, the mechanisms of coding and transferring information by these functionalities are not yet complete understood, mainly because of (i)the complex physiological microenvironment in which GAGs interactions occur and (ii)the inability to access homogeneous GAGs[2]. In this work, we propose model surfaces bearing GAGs with different sulfation degree as platform to investigate the pathways by which mesenchymal stem cells (MSCs) sense and respond to this peculiar functionality: the -OSO3H. We have selected two natural GAGs for this study: hyaluronic acid (HA) because it is the only non-sulfated glycan and heparin (HEP) as it is the GAG with the highest degree of sulfation. To obtain a larger range of sulfation degrees, we have also prepared a synthetic analogue of HA with a sulfation degree of 1.4 (sHA). All these GAGs were covalently bonded to aminothiols deposited on gold surfaces. MSCs, both from bone marrow and adipose tissue, adhered well to all surfaces. Formation of focal adhesions was observed after only 1h of culture for bone marrow derived MSCs regardless the used substrate. The presence of –OSO3H groups induced different morphology and cytoskeleton organisation: formation of longer filopodia and well pronounced actin fibers were visible for the MSCs from both sources. Moreover, cells were more spread after 24h in contact with – OSO3H containing surfaces. Cells behaved similarly on both sulfated surfaces (sHA and HEP) and differences in cell morphology were less obvious: higher sulfation degree induced less lamellipodia formation while filopodia number and length increased. In summary, the present study provides evidence that sulfation degree of GAGs triggers distinct cytoskeleton organisation in mesenchymal stem cells that may be related with the differentiation of those cells. However, further studies at the molecular level about the exact mechanism of these processes need to be carried out.

On behalf of a UK-based upmarket retailer, Mitie Asset Investment (Mitie) has purchased two 4-engine updraft gasification combined heat and power (CHP) plants from Stirling.DK (SDK). The CHP plants are to be part of a trigeneration system delivering heat, electricity and cooling to provide a complete carbon-neutral energy solution for UK supermarkets. Each SDK plant will deliver 140 kWe and 500 kW of useful heating (600 kW total heating), using app. 320 kg of fresh wood chips per hour (800 kW input), operating 6,565 hours a year. The aim of the paper is to provide an overview of technical and economic benefits of utilizing biofuelled trigeneration systems in the retail sector. We expect that the largest gains will come from the utilization of heat in an absorption/adsorption chilling system to substitute cooling from electricity. To the authors' knowledge, there have been no previous applications of small-scale solid biofuel combustion systems for trigeneration in the retail sector. These two cases should provide valuable information on the commercial viability of small-scale biofuel systems., Proceedings of the 20th European Biomass Conference and Exhibition, 18-22 June 2012, Milan, Italy, pp. 1801-1807

Investment cases are prepared for a Stirling¬powered wood chip combined heat and power plant with an electrical capacity of 140 kW and a thermal capacity of 560 kW, for a general case in Germany, the UK and Denmark. Existing national financial support schemes are included for each of the three countries. The impact of the support schemes on the net present value in each case is assessed. Differences between the three countries are discussed, especially with regard to differences in national energy prices, renewable energy ambitions and existing energy profiles., Proceedings of the 20th European Biomass Conference and Exhibition, 18-22 June 2012, Milan, Italy, pp. 2172-2180

A novel test battery consisting of self-assessments and motor tests (tapping and spiral drawing) for patients with Parkinson’s disease (PD) was developed for a hand computer with touch screen in a telemedicine setting. Tests are performed four times per day in the home environment during weeklong test periods. Results are processed into scores for different dimensions of the symptom state and an ‘overall score’ reflecting the global condition of a patient during a test period. The test battery was validated in a separate study recently submitted to Mov Disord. This test battery is currently being used in an open longitudinal trial (DAPHNE, EudraCT No. 2005- 002654-21) by sixty-five patients with advanced PD at nine clinics around Sweden. On inclusion, the patients were either receiving treatment with duodenal levodopa/carbidopa infusion (Duodopa®) (n=36), or they were candidates for receiving this treatment (n=29). We now present interim results for the first twelve months. Test periods were performed in three-month intervals. During most of the periods, UPDRS ratings were performed in afternoons at the start of the week. In twenty of the patients, scores were available during individually optimized oral polypharamacy, before receiving infusion and at least one test period after having started infusion treatment. Usability and compliance with performing tests, this far are good, both with patients and clinical staff. Correlations between test periods 2 and 3 during infusion treatment (three months apart) are stronger for overall test score than for total UPDRS, indicating good reliability. The correlation between overall test score and UPDRS for all test periods is adequate (r=-0.6). In an exact Wilcoxon signed rank test, where the endpoint is the change from the first to the twelve month test period (n=25), there was no change in test results in any of the test battery dimensions for the patients already receiving infusion when included. However, in the patients entering the study before receiving infusion, there was a significant change (improvement) from the baseline to the twelve month test period in dimensions; ‘off’, ‘dyskinesia’ and ‘satisfied’ and in the ‘overall score’ (n=15). The mean improvement in overall score after infusion was 29% (p=0.015). We conclude that the test battery is able to measure a functional improvement with infusion that is sustained over at least twelve months.

Objective: To compare results from various tapping tests with diary responses in advanced PD. Background: A home environment test battery for assessing patient state in advanced PD, consisting of diary assessments and motor tests was constructed for a hand computer with touch screen and mobile communication. The diary questions: 1. walking, 2. time in off , on and dyskinetic states, 3. off at worst, 4. dyskinetic at worst, 5. cramps, and 6. satisfied with function, relate to the recent past. Question 7, self-assessment, allows seven steps from -3 ( very off ) to +3 ( very dyskinetic ) and relate to right now. Tapping tests outline: 8. Alternately tapping two fields (un-cued) with right hand 9. Same as 8 but using left hand 10. Tapping an active field (out of two) following a system-generated rhythm (increasing speed) with the dominant hand 11. Tapping an active field (out of four) that randomly changes location when tapped using the dominant hand Methods: 65 patients (currently on Duodopa, or candidates for this treatment) entered diary responses and performed tapping tests four times per day during one to six periods of seven days length. In total there were 224 test periods and 6039 test occasions. Speed for tapping test 10 was discardedand tests 8 and 9 were combined by taking means. Descriptive statistics were used to present the variation of the test variables in relation to self assessment (question 7). Pearson correlation coefficients between speed and accuracy (percent correct) in tapping tests and diary responses were calculated. Results: Mean compliance (percentage completed test occasions per test period) was 83% and the median was 93%. There were large differences in both mean tapping speed and accuracy between the different self-assessed states. Correlations between diary responses and tapping results were small (-0.2 to 0.3, negative values for off-time and dyskinetic-time that had opposite scale directions). Correlations between tapping results were all positive (0.1 to 0.6). Conclusions: The diary responses and tapping results provided different information. The low correlations can partly be explained by the fact that questions related to the past and by random variability, which could be reduced by taking means over test periods. Both tapping speed and accuracy reflect the motor function of the patient to a large extent.

Objective: We present a new evaluation of levodopa plasma concentrations and clinical effects during duodenal infusion of a levodopa/carbidopa gel (Duodopa ) in 12 patients with advanced Parkinson s disease (PD), from a study reported previously (Nyholm et al, Clin Neuropharmacol 2003; 26(3): 156-163). One objective was to investigate in what state of PD we can see the greatest benefits with infusion compared with corresponding oral treatment (Sinemet CR). Another objective was to identify fluctuating response to levodopa and correlate to variables related to disease progression. Methods: We have computed mean absolute error (MAE) and mean squared error (MSE) for the clinical rating from -3 (severe parkinsonism) to +3 (severe dyskinesia) as measures of the clinical state over the treatment periods of the study. Standard deviation (SD) of the rating was used as a measure of response fluctuations. Linear regression and visual inspection of graphs were used to estimate relationships between these measures and variables related to disease progression such as years on levodopa (YLD) or unified PD rating scale part II (UPDRS II).Results: We found that MAE for infusion had a strong linear correlation to YLD (r2=0.80) while the corresponding relation for oral treatment looked more sigmoid, particularly for the more advanced patients (YLD>18).