Recommendations for in vitro evaluation of blood components collected, prepared and stored in non-DEHP medical devices.

BACKGROUND AND OBJECTIVES: DEHP, di(2-ethylhexyl) phthalate, is the most common member of the class of ortho-phthalates, which are used as plasticizers. The Medical Device Regulation has restricted the use of phthalates in medical devices. Also DEHP has been added to the Annex XIV of REACH, "Registration, Evaluation, Authorisation and Restriction of Chemicals" due to its endocrine disrupting properties to the environment. As such, the sunset date for commercialisation of DEHP-containing blood bags is May 27th 2025. There are major concerns in meeting this deadline as these systems have not yet been fully validated and/or CE-marked. Also, since DEHP is known to affect red cell quality during storage, it is imperative to transit to non-DEHP without affecting blood product quality. Here, EBA members aim to establish common grounds on the evaluation and assessment of blood components collected, prepared and stored in non-DEHP devices. MATERIALS AND METHODS: Based on data as well as the input of relevant stakeholders a rationale for the validation of each component was composed. RESULTS: The red cell components will require the most extensive validation as their quality is directly affected by the absence of DEHP, as opposed to platelet and plasma components. CONCLUSION: Studies in the scope of evaluating the quality of blood products obtained with non-DEHP devices, under the condition that they are carried out according to these recommendations, could be used by all members of the EBA to serve as scientific support in the authorization process specific to their jurisdiction or for their internal validation use.

A comparison of platelet quality between platelets from healthy donors and hereditary hemochromatosis donors over seven-day storage.

BACKGROUND: Therapeutic phlebotomy is the standard treatment of hereditary hemochromatosis (HH), the most common genetic disease in people of Northern European descent. Red cell concentrates from HH donors have been reported safe for transfusion, but little data is available on the storage properties of platelet concentrates from HH donors. STUDY DESIGN AND METHODS: Whole blood was collected from 10 healthy individuals and 10 newly diagnosed HH patients with elevated serum ferritin. Platelet-rich plasma (PRP) was prepared and split into four 20-mL units. Platelet quality tests were performed on days 0, 1, 3, 5, and 7 of storage, including platelet aggregation (ADP, arachidonic acid, collagen, and epinephrine agonists), blood gas analysis, flow cytometry (CD41, CD42b, and CD62P expression), and ELISA (sCD40L and sCD62p in supernatant). RESULTS: Mean serum ferritin levels were higher in HH patients than in controls (847.5 vs 45.8 ng/mL, P < .001). Overall, no difference in quality test results was observed between the two study groups over 7-day storage (P > .05), including blood gas analysis, platelet aggregation, and expression of surface (CD62p and CD42b) and secreted (sCD62P and sCD40L) activation markers. Expected alterations in metabolic (CO2 and glucose decrease, O2 and lactate increase, P < .001) and platelet activation markers (CD42b decrease, CD62P increase, P < .05) over time were observed in both groups. CONCLUSION: Although these findings indicate that platelets of individuals with HH are comparable to platelets from healthy donors, more extensive studies are needed before definite conclusions can be drawn.

Visualizing metabolic network dynamics through time-series metabolomic data.

BACKGROUND: New technologies have given rise to an abundance of -omics data, particularly metabolomic data. The scale of these data introduces new challenges for the interpretation and extraction of knowledge, requiring the development of innovative computational visualization methodologies. Here, we present GEM-Vis, an original method for the visualization of time-course metabolomic data within the context of metabolic network maps. We demonstrate the utility of the GEM-Vis method by examining previously published data for two cellular systems-the human platelet and erythrocyte under cold storage for use in transfusion medicine. RESULTS: The results comprise two animated videos that allow for new insights into the metabolic state of both cell types. In the case study of the platelet metabolome during storage, the new visualization technique elucidates a nicotinamide accumulation that mirrors that of hypoxanthine and might, therefore, reflect similar pathway usage. This visual analysis provides a possible explanation for why the salvage reactions in purine metabolism exhibit lower activity during the first few days of the storage period. The second case study displays drastic changes in specific erythrocyte metabolite pools at different times during storage at different temperatures. CONCLUSIONS: The new visualization technique GEM-Vis introduced in this article constitutes a well-suitable approach for large-scale network exploration and advances hypothesis generation. This method can be applied to any system with data and a metabolic map to promote visualization and understand physiology at the network level. More broadly, we hope that our approach will provide the blueprints for new visualizations of other longitudinal -omics data types. The supplement includes a comprehensive user's guide and links to a series of tutorial videos that explain how to prepare model and data files, and how to use the software SBMLsimulator in combination with further tools to create similar animations as highlighted in the case studies.

Metabolomics study of platelet concentrates photochemically treated with amotosalen and UVA light for pathogen inactivation.

BACKGROUND: The risk of bacterial contamination and the deterioration of platelet (PLT) quality limit the shelf-life of platelet concentrates (PCs). The INTERCEPT pathogen inactivation system reduces the risk of pathogen transmission by inhibiting nucleic acid replication using a combination of a photo-reactive compound and UVA illumination. The goal of this study was to investigate the effects the INTERCEPT system has on the PLT metabolome and metabolic activity. STUDY DESIGN AND METHODS: Paired units of buffy coat-derived PCs were generated using a pool and split strategy (n = 8). The paired PCs were either treated with the INTERCEPT system or left untreated. Samples were collected on Days 1, 2, 4, and 7 of storage. Ultra-performance chromatography coupled with time-of-flight mass spectrometry was used to analyze the extra- and intracellular metabolomes. Constraint-based metabolic modeling was then used to predict the metabolic activity of the stored PLTs. RESULTS: A relatively large number of metabolites in the extracellular environment were depleted during the processing steps of the INTERCEPT system, in particular, metabolites with hydrophobic functional groups, including acylcarnitines and lysophosphatidylcholines. In the intracellular environment, alterations in glucose and glycerophospholipid metabolism and decreased levels of 2-hydroxyglutarate were observed following the INTERCEPT treatment. Untargeted metabolomics analysis revealed residual amotosalen dimers present in the treated PCs. Systems-level analysis of PLT metabolism indicated that the INTERCEPT system does not have a significant impact on the PLT energy metabolism and nutrient utilization. CONCLUSIONS: The INTERCEPT system significantly alters the metabolome of the stored PCs without significantly influencing PLT energy metabolism.

Pathogen inactivation with amotosalen plus UVA illumination minimally impacts microRNA expression in platelets during storage under standard blood banking conditions.

BACKGROUND: To reduce the risk of transfusion transmission infection, nucleic acid targeted methods have been developed to inactivate pathogens in PCs. miRNAs have been shown to play an important role in platelet function, and changes in the abundance of specific miRNAs during storage have been observed, as have perturbation effects related to pathogen inactivation (PI) methods. The aim of this work was to investigate the effects of PI on selected miRNAs during storage. STUDY DESIGN AND METHODS: Using a pool and split strategy, 3 identical buffy coat PC units were generated from a pool of 24 whole blood donors. Each unit received a different treatment: 1) Untreated platelet control in platelet additive solution (C-PAS); 2) Amotosalen-UVA-treated platelets in PAS (PI-PAS); and 3) untreated platelets in donor plasma (U-PL). PCs were stored for 7 days under standard blood banking conditions. Standard platelet quality control (QC) parameters and 25 selected miRNAs were analyzed. RESULTS: During the 7-day storage period, differences were found in several QC parameters relating to PI treatment and storage in plasma, but overall the three treatments were comparable. Out of 25 miRNA tested changes in regulation of 5 miRNA in PI-PAS and 3 miRNA U-PL where detected compared to C-PAS. A statistically significant difference was observed in down regulations miR-96-5p on Days 2 and 4, 61.9% and 61.8%, respectively, in the PI-PAS treatment. CONCLUSION: Amotosalen-UVA treatment does not significantly alter the miRNA profile of platelet concentrates generated and stored using standard blood banking conditions.

Systems analysis of metabolism in platelet concentrates during storage in platelet additive solution.

Platelets (PLTs) deteriorate over time when stored within blood banks through a biological process known as PLT storage lesion (PSL). Here, we describe the refinement of the biochemical model of PLT metabolism, iAT-PLT-636, and its application to describe and investigate changes in metabolism during PLT storage. Changes in extracellular acetate and citrate were measured in buffy coat and apheresis PLT units over 10 days of storage in the PLT additive solution T-Sol. Metabolic network analysis of these data was performed alongside our prior metabolomics data to describe the metabolism of fresh (days 1-3), intermediate (days 4-6), and expired (days 7-10) PLTs. Changes in metabolism were studied by comparing metabolic model flux predictions of iAT-PLT-636 between stages and between collection methods. Extracellular acetate and glucose contribute most to central carbon metabolism in PLTs. The anticoagulant citrate is metabolized in apheresis-stored PLTs and is converted into aconitate and, to a lesser degree, malate. The consumption of nutrients changes during storage and reflects altered PLT activation profiles following their collection. Irrespective of the collection method, a slowdown in oxidative phosphorylation takes place, consistent with mitochondrial dysfunction during PSL. Finally, the main contributors to intracellular ammonium and NADPH are highlighted. Future optimization of flux through these pathways provides opportunities to address intracellular pH changes and reactive oxygen species, which are both of importance to PSL. The metabolic models provide descriptions of PLT metabolism at steady state and represent a platform for future PLT metabolic research.

Quantitative time-course metabolomics in human red blood cells reveal the temperature dependence of human metabolic networks.

The temperature dependence of biological processes has been studied at the levels of individual biochemical reactions and organism physiology (e.g. basal metabolic rates) but has not been examined at the metabolic network level. Here, we used a systems biology approach to characterize the temperature dependence of the human red blood cell (RBC) metabolic network between 4 and 37 °C through absolutely quantified exo- and endometabolomics data. We used an Arrhenius-type model (Q10) to describe how the rate of a biochemical process changes with every 10 °C change in temperature. Multivariate statistical analysis of the metabolomics data revealed that the same metabolic network-level trends previously reported for RBCs at 4 °C were conserved but accelerated with increasing temperature. We calculated a median Q10 coefficient of 2.89 ± 1.03, within the expected range of 2-3 for biological processes, for 48 individual metabolite concentrations. We then integrated these metabolomics measurements into a cell-scale metabolic model to study pathway usage, calculating a median Q10 coefficient of 2.73 ± 0.75 for 35 reaction fluxes. The relative fluxes through glycolysis and nucleotide metabolism pathways were consistent across the studied temperature range despite the non-uniform distributions of Q10 coefficients of individual metabolites and reaction fluxes. Together, these results indicate that the rate of change of network-level responses to temperature differences in RBC metabolism is consistent between 4 and 37 °C. More broadly, we provide a baseline characterization of a biochemical network given no transcriptional or translational regulation that can be used to explore the temperature dependence of metabolism.

Biomarkers defining the metabolic age of red blood cells during cold storage.

Metabolomic investigations of packed red blood cells (RBCs) stored under refrigerated conditions in saline adenine glucose mannitol (SAGM) additives have revealed the presence of 3 distinct metabolic phases, occurring on days 0-10, 10-18, and after day 18 of storage. Here we used receiving operating characteristics curve analysis to identify biomarkers that can differentiate between the 3 metabolic states. We first recruited 24 donors and analyzed 308 samples coming from RBC concentrates stored in SAGM and additive solution 3. We found that 8 extracellular compounds (lactic acid, nicotinamide, 5-oxoproline, xanthine, hypoxanthine, glucose, malic acid, and adenine) form the basis for an accurate classification/regression model and are able to differentiate among the metabolic phases. This model was then validated by analyzing an additional 49 samples obtained by preparing 7 new RBC concentrates in SAGM. Despite the technical variability associated with RBC processing strategies, verification of these markers was independently confirmed in 2 separate laboratories with different analytical setups and different sample sets. The 8 compounds proposed here highly correlate with the metabolic age of packed RBCs, and can be prospectively validated as biomarkers of the RBC metabolic lesion.

To Wash or Not to Wash? Comparison of Patient Outcome after Infusion of Cryopreserved Autologous Hematopoietic Stem Cells before and after the Replacement of Manual Washing by Bedside Thawing.

BACKGROUND: Prior to infusion, cryopreserved autologous peripheral blood stem cell (auto-PBSC) grafts can either be thawed at the bedside or thawed and washed at the laboratory. At our center, manual washing of grafts prior to infusion was discontinued in April 2012 and bedside thawing was implemented. METHODS: This study compares the outcomes of two patient groups who received auto-PBSC either after post-thaw washing (n = 84) or bedside thawing (n = 83). RESULTS: No life-threatening infusion-related side effects were reported in either group. There was no significant difference in the mean CD34+ cells/kg dose of infused auto-PBSC in the two groups (p = 0.41), nor in the number of days to neutrophils > 0.5 × 10(9)/L (p = 0.14), days to platelets > 20 × 10(9)/L (p = 0.64), or days to platelets > 50 × 10(9)/L (p = 0.62) after transplant. There was also no difference in the number of days on total parenteral nutrition (p = 0.69), days on G-CSF therapy (p = 0.48), or days with fever (p = 0.73). Finally, there was no significant difference in the number of red cell units transfused (p = 0.32), or platelet units transfused (p = 0.94) after the transplant. One-hundred-day mortality was identical in the two groups (2.4%). CONCLUSION: Both thawing procedures are safe and result in acceptable engraftment and patient outcomes.

Mannose and fructose metabolism in red blood cells during cold storage in SAGM.

BACKGROUND: Alternate sugar metabolism during red blood cell (RBC) storage is not well understood. Here we report fructose and mannose metabolism in RBCs during cold storage in SAGM and the impact that these monosaccharides have on metabolic biomarkers of RBC storage lesion. STUDY DESIGN AND METHODS: RBCs were stored in SAGM containing uniformly labeled 13 C-fructose or 13 C-mannose at 9 or 18 mmol/L concentration for 25 days. RBCs and media were sampled at 14 time points during storage and analyzed using ultraperformance liquid chromatography-mass spectrometry. Blood banking quality assurance measurements were performed. RESULTS: Red blood cells incorporated fructose and mannose during cold storage in the presence of glucose. Mannose was metabolized in preference to glucose via glycolysis. Fructose lowered adenosine triphosphate (ATP) levels and contributed little to ATP maintenance when added to SAGM. Both monosaccharides form the advanced glycation end product glycerate. Mannose activates enzymes in the RBC that take part in glycan synthesis. CONCLUSIONS: Fructose or mannose addition to RBC SAGM concentrates may not offset the shift in metabolism of RBCs that occurs after 10 days of storage. Fructose and mannose metabolism at 4°C in SAGM reflects their metabolism at physiologic temperature. Glycerate excretion is a measure of protein deglycosylation activity in stored RBCs. No cytoprotective effect was observed upon the addition of either fructose or mannose to SAGM.

Metabolic fate of adenine in red blood cells during storage in SAGM solution.

BACKGROUND: Red blood cells (RBCs) are routinely stored and transfused worldwide. Recently, metabolomics have shown that RBCs experience a three-phase metabolic decay process during storage, resulting in the definition of three distinct metabolic phenotypes, occurring between Days 1 and 10, 11 and 17, and 18 and 46. Here we use metabolomics and stable isotope labeling analysis to study adenine metabolism in RBCs. STUDY DESIGN AND METHODS: A total of 6 units were prepared in SAGM or modified additive solutions (ASs) containing 15 N5 -adenine. Three of them were spiked with 15 N5 -adenine on Days 10, 14, and 17 during storage. Each unit was sampled 10 times spanning Day 1 to Day 32. At each time point metabolic profiling was performed. RESULTS: We increased adenine concentration in the AS and we pulsed the adenine concentration during storage and found that in both cases the RBCs' main metabolic pathways were not affected. Our data clearly show that RBCs cannot consume adenine after 18 days of storage, even if it is still present in the storage solution. However, increased levels of adenine influenced S-adenosylmethionine metabolism. CONCLUSION: In this work, we have studied in detail the metabolic fate of adenine during RBC storage in SAGM. Adenine is one of the main substrates used by RBCs, but the metabolic shift observed during storage is not caused by an absence of adenine later in storage. The rate of adenine consumption strongly correlated with duration of storage but not with the amount of adenine present in the AS.

Identified metabolic signature for assessing red blood cell unit quality is associated with endothelial damage markers and clinical outcomes.

BACKGROUND: There has been interest in determining whether older red blood cell (RBC) units have negative clinical effects. Numerous observational studies have shown that older RBC units are an independent factor for patient mortality. However, recently published randomized clinical trials have shown no difference of clinical outcome for patients receiving old or fresh RBCs. An overlooked but essential issue in assessing RBC unit quality and ultimately designing the necessary clinical trials is a metric for what constitutes an old or fresh RBC unit. STUDY DESIGN AND METHODS: Twenty RBC units were profiled using quantitative metabolomics over 42 days of storage in SAGM with 3- to 4-day time intervals. Metabolic pathway usage during storage was assessed using systems biology methods. The detected time intervals of the metabolic states were compared to clinical outcomes. RESULTS: Using multivariate statistics, we identified a nonlinear decay process exhibiting three distinct metabolic states (Days 0-10, 10-17, and 17-42). Hematologic variables traditionally measured in the transfusion setting (e.g., pH, hemolysis, RBC indices) did not distinguish these three states. Systemic changes in pathway usage occurred between the three states, with key pathways changing in both magnitude and direction. Finally, an association was found between the time periods of the metabolic states with the clinical outcomes of more than 280,000 patients in the country of Denmark transfused over the past 15 years and endothelial damage markers in healthy volunteers undergoing autologous transfusions. CONCLUSION: The state of RBC metabolism may be a better indicator of cellular quality than traditional hematologic variables.

Metabolomic analysis of platelets during storage: a comparison between apheresis- and buffy coat-derived platelet concentrates.

BACKGROUND: Platelet concentrates (PCs) can be prepared using three methods: platelet (PLT)-rich plasma, apheresis, and buffy coat. The aim of this study was to obtain a comprehensive data set that describes metabolism of buffy coat-derived PLTs during storage and to compare it with a previously published parallel data set obtained for apheresis-derived PLTs. STUDY DESIGN AND METHODS: During storage we measured more than 150 variables in 8 PLT units, prepared by the buffy coat method. Samples were collected at seven different time points resulting in a data set containing more than 8000 measurements. This data set was obtained by combining a series of standard quality control assays to monitor the quality of stored PLTs and a deep coverage metabolomics study using liquid chromatography coupled with mass spectrometry. RESULTS: Stored PLTs showed a distinct metabolic transition occurring 4 days after their collection. The transition was evident in PLT produced by both production methods. Apheresis-derived PLTs showed a clearer phenotype of PLT activation during early days of storage. The activated phenotype of apheresis PLTs was accompanied by a higher metabolic activity, especially related to glycolysis and the tricarboxylic acid cycle. Moreover, the extent of the activation differed between bags resulting in interbag variability in the storage lesion of apheresis-prepared PLTs. This may be related to donor-related polymorphism. CONCLUSION: This study demonstrated two discrete metabolic phenotypes in stored PLTs prepared with both apheresis and buffy coat methods. PLT activation occurs during the first metabolic phenotype and might lead to a low reproducibility of the apheresis PCs.

Impact of chain length on antibacterial activity and hemocompatibility of quaternary N-alkyl and n,n-dialkyl chitosan derivatives.

A highly efficient method for chemical modification of chitosan biopolymers by reductive amination to yield N,N-dialkyl chitosan derivatives was developed. The use of 3,6-O-di-tert-butyldimethylsilylchitosan as a precursor enabled the first 100% disubstitution of the amino groups with long alkyl chains. The corresponding mono N-alkyl derivatives were also synthesized, and all the alkyl compounds were then quaternized using an optimized procedure. These well-defined derivatives were studied for antibacterial activity against Gram positive S. aureus, E. faecalis, and Gram negative E. coli, P. aeruginosa, which could be correlated to the length of the alkyl chain, but the order was dependent on the bacterial strain. Toxicity against human red blood cells and human epithelial Caco-2 cells was found to be proportional to the length of the alkyl chain. The most active chitosan derivatives were found to be more selective for killing bacteria than the quaternary ammonium disinfectants cetylpyridinium chloride and benzalkonium chloride, as well as the antimicrobial peptides melittin and LL-37.

Comprehensive metabolomic study of platelets reveals the expression of discrete metabolic phenotypes during storage.

BACKGROUND: Platelet (PLT) concentrates are routinely stored for 5 to 7 days. During storage they exhibit what has been termed PLT storage lesion (PSL), which is evident by a loss of hemostatic function when transfused into patients. The overall goal of this study was to obtain a comprehensive data set describing PLT metabolism during storage. STUDY DESIGN AND METHODS: The experimental approach adopted to achieve this goal combined a series of standard assays to monitor the quality of stored PLTs and a deep-coverage metabolomics study using liquid chromatography coupled with mass spectrometry performed on both the extracellular and the intracellular environments. During storage we measured 174 different variables in 6 PLT units, collected by apheresis. Samples were collected at eight different time points resulting in a data set containing more than 8000 measurements. RESULTS: Stored PLTs did not undergo a monotonic decay, but experienced systematic changes in metabolism reflected in three discrete metabolic phenotypes: The first (Days 0-3) was associated with active glycolysis, pentose phosphate pathway, and glutathione metabolism and down regulation of tricarboxylic acid (TCA) cycle. The second (Days 4-6) was associated with a more active TCA cycle as well as increased purine metabolism. A third metabolic phenotype of less clinical relevance (Days 7-10) was associated with a faster decay of cellular metabolism. CONCLUSION: PSL is not associated with a linear decay of metabolism, but rather with successive metabolic shifts. These findings may give new insight into the mechanisms underlying PSL and encourage the deployment of systems biology methods to PSL.

The effect of substituent, degree of acetylation and positioning of the cationic charge on the antibacterial activity of quaternary chitosan derivatives.

A series of water-soluble cationic chitosan derivatives were prepared by chemoselective functionalization at the amino group of five different parent chitosans having varying degrees of acetylation and molecular weight. The quaternary moieties were introduced at different alkyl spacer lengths from the polymer backbone (C-0, C-2 and C-6) with the aid of 3,6-di-O-tert-butyldimethylsilyl protection of the chitosan backbone, thus allowing full (100%) substitution of the free amino groups. All of the derivatives were characterized using 1H-NMR, 1H-1H COSY and FT-IR spectroscopy, while molecular weight was determined by GPC. Antibacterial activity was investigated against Gram positive S. aureus and Gram negative E. coli. The relationship between structure and activity/toxicity was defined, considering the effect of the cationic group's structure and its distance from the polymer backbone, as well as the degree of acetylation within a molecular weight range of 7-23 kDa for the final compounds. The N,N,N-trimethyl chitosan with 100% quaternization showed the highest antibacterial activity with moderate cytotoxicity, while increasing the spacer length reduced the activity. Trimethylammoniumyl quaternary ammonium moieties contributed more to activity than 1-pyridiniumyl moieties. In general, no trend in the antibacterial activity of the compounds with increasing molecular weight or degree of acetylation up to 34% was observed.

Temperature Dependence of Platelet Metabolism.

Temperature plays a fundamental role in biology, influencing cellular function, chemical reaction rates, molecular structures, and interactions. While the temperature dependence of many biochemical reactions is well defined in vitro, the effect of temperature on metabolic function at the network level is poorly understood, and it remains an important challenge in optimizing the storage of cells and tissues at lower temperatures. Here, we used time-course metabolomic data and systems biology approaches to characterize the effects of storage temperature on human platelets (PLTs) in a platelet additive solution. We observed that changes to the metabolome with storage time do not simply scale with temperature but instead display complex temperature dependence, with only a small subset of metabolites following an Arrhenius-type relationship. Investigation of PLT energy metabolism through integration with computational modeling revealed that oxidative metabolism is more sensitive to temperature changes than glycolysis. The increased contribution of glycolysis to ATP turnover at lower temperatures indicates a stronger glycolytic phenotype with decreasing storage temperature. More broadly, these results demonstrate that the temperature dependence of the PLT metabolic network is not uniform, suggesting that efforts to improve the health of stored PLTs could be targeted at specific pathways.

Partially acetylated chitooligosaccharides bind to YKL-40 and stimulate growth of human osteoarthritic chondrocytes.

Recent evidences indicating that cellular kinase signaling cascades are triggered by oligomers of N-acetylglucosamine (ChOS) and that condrocytes of human osteoarthritic cartilage secrete the inflammation associated chitolectin YKL-40, prompted us to study the binding affinity of partially acetylated ChOS to YKL-40 and their effect on primary chondrocytes in culture. Extensive chitinase digestion and filtration of partially deacetylated chitin yielded a mixture of ChOS (Oligomin™) and further ultrafiltration produced T-ChOS™, with substantially smaller fraction of the smallest sugars. YKL-40 binding affinity was determined for the different sized homologues, revealing micromolar affinities of the larger homologues to YKL-40. The response of osteoarthritic chondrocytes to Oligomin™ and T-ChOS™ was determined, revealing 2- to 3-fold increases in cell number. About 500 µg/ml was needed for Oligomin™ and around five times lower concentration for T-ChOS™, higher concentrations abolished this effect for both products. Addition of chitotriose inhibited cellular responses mediated by larger oligosaccharides. These results, and the fact that the partially acetylated T-ChOS™ homologues should resist hydrolysis, point towards a new therapeutic concept for treating inflammatory joint diseases.

Solution casting of chitosan membranes for in vitro evaluation of bioactivity.

BACKGROUND: Considerable research is focusing on the surface modification of titanium implants for the treatment of orthopaedic tissue injuries to increase the success of orthopaedic fixations. Chitosan is one of the natural materials under investigation based on several favourable properties. Numerous techniques have been described for the preparation of chitosan membranes, including solution casting methods for the investigation of bioactivity before applying coatings onto potential titanium implants. Solution casting enables the easy in-house evaluation of chitosan membranes and allows for the selection of promising chitosan materials. RESULTS: We present a method for the standardized and easily applied preparation of chitosan membranes by solution casting. This protocol is suitable for chitosan materials spanning a wide degree of deacetylation, being derived from different chitin sources and chitosan derivatives with novel properties. We detail the preparation and quality control methods in order to prepare membranes with favourable bioactivity, sustaining cell attachment and proliferation for extended culture periods. CONCLUSIONS: The possibilities associated with the use of chitosan in tissue engineering applications are far from being exhausted and numerous challenges remain prior to successful translation into the clinics. Based on our experience, we have developed simple in-house methods for quality control of homogeneous membrane casting and early prediction of successful experimental outcome.

Structure-activity relationship for antibacterial chitosan carrying cationic and hydrophobic moieties.

To overcome the problem of antibiotic resistance and toxicity of synthetic polymers, herein we report the synthesis of biocompatible polymers which can serve as broad spectrum antimicrobials. A regioselective synthetic method was developed to synthesize N-functionalized chitosan polymers having similar degree of substitution of cationic and hydrophobic functionality with different lipophilic chains. We obtained optimum antibacterial effect by utilizing the combination of cationic and longer lipophilic chain in the polymer, against four bacterial strains. Inhibition and killing of bacteria were more pronounced in Gram positive bacteria than in Gram negative bacteria. Growth kinetics and scanning electron microscopy imaging of the polymer treated bacterial cells confirmed the inhibition of bacterial growth, morphological changes in the structure and membrane disruption in the cells as compared to the growth control for each strain. Further investigation into the toxicity and selectivity of the polymers guided us to develop a structure-activity relationship for this class of biocompatible polymers.