A novel on-a-chip system with a 3D-bioinspired gut mucus suitable to investigate bacterial endotoxins dynamics.

The possible pathogenic impact of pro-inflammatory molecules produced by the gut microbiota is one of the hypotheses considered at the basis of the biomolecular dialogue governing the microbiota-gut-brain axis. Among these molecules, lipopolysaccharides (LPS) produced by Gram-negative gut microbiota strains may have a potential key role due to their toxic effects in both the gut and the brain. In this work, we engineered a new dynamic fluidic system, the MINERVA device (MI-device), with the potential to advance the current knowledge of the biological mechanisms regulating the microbiota-gut molecular crosstalk. The MI-device supported the growth of bacteria that are part of the intestinal microbiota under dynamic conditions within a 3D moving mucus model, with features comparable to the physiological conditions (storage modulus of 80 ± 19 Pa, network mesh size of 41 ± 3 nm), without affecting their viability (∼ 109 bacteria/mL). The integration of a fluidically optimized and user-friendly design with a bioinspired microenvironment enabled the sterile extraction and quantification of the LPS produced within the mucus by bacteria (from 423 ± 34 ng/mL to 1785 ± 91 ng/mL). Compatibility with commercially available Transwell-like inserts allows the user to precisely control the transport phenomena that occur between the two chambers by selecting the pore density of the insert membrane without changing the design of the system. The MI-device is able to provide the flow of sterile medium enriched with LPS directly produced by bacteria, opening up the possibility of studying the effects of bacteria-derived molecules on cells in depth, as well as the assessment and characterization of their effects in a physiological or pathological scenario.

Engineering biological gradients.

Biological gradients profoundly influence many cellular activities, such as adhesion, migration, and differentiation, which are the key to biological processes, such as inflammation, remodeling, and tissue regeneration. Thus, engineered structures containing bioinspired gradients can not only support a better understanding of these phenomena, but also guide and improve the current limits of regenerative medicine. In this review, we outline the challenges behind the engineering of devices containing chemical-physical and biomolecular gradients, classifying them according to gradient-making methods and the finalities of the systems. Different manufacturing processes can generate gradients in either in-vitro systems or scaffolds, which are suitable tools for the study of cellular behavior and for regenerative medicine; within these, rapid prototyping techniques may have a huge impact on the controlled production of gradients. The parallel need to develop characterization techniques is addressed, underlining advantages and weaknesses in the analysis of both chemical and physical gradients.

Evaluating outcome of prophylaxis in haemophilia: objective and self-reported instruments should be combined.

INTRODUCTION: Routine outcome assessment of prophylaxis should use validated tools, while balancing comprehensiveness and burden. Collecting overlapping information should be avoided. AIM: To assess correlations between different outcome assessment tools in haemophilia. METHODS: From an international cross-sectional study, data on objective outcome (Haemophilia Joint Health Score (HJHS 2.1, range 0-124), radiological Pettersson score) and self-reported joint bleeding, Haemophilia Activities List (HAL, range 100-0), health-related quality of life (SF-36, including five physical and five mental domain scores, range 100-0), and Utility (SF6D and EQ-5D, range 1.0-0) were extracted. Spearman's correlations were calculated: ≥0.8 very strong, 0.60-0.79 strong, 0.40-0.59 moderate. RESULTS: Ninety patients with severe haemophilia, on prophylaxis since median age 3.4 years, were evaluated at median 25.5 years (range 16.0-37.6). Objective outcome was favourable (median HJHS 2.1 6 points, Pettersson score 9 points). Self-reported outcome showed a median of 7 joint bleeds in 5 years, median HAL sum 96 points, high scores for physical domains of SF-36 (median 80-95) and high Utility values (median SF6D 0.87; EQ-5D 0.84). Physical examination (HJHS 2.1) showed strong correlation with radiological scores, moderate correlation with physical domains of the SF-36 and Utility, but no correlation with self-reported bleeding or limitations in activities (HAL). Bleeding was not associated with any other outcome parameter. The HAL was only correlated with the SF36 'Physical functioning' domain. CONCLUSION: For the evaluation of patients on early prophylaxis, information on bleeding should be complemented by objective joint assessment as well as self-reported limitations in activities and quality of life.

Using routine Haemophilia Joint Health Score for international comparisons of haemophilia outcome: standardization is needed.

INTRODUCTION: Haemophilia Joint Health Score (HJHS) is the most sensitive validated score for physical examination of joint health in haemophilia. HJHS performed at regular intervals can be used for clinical monitoring as well as for comparative outcomes research. AIM: To determine whether routinely collected HJHS could be used to compare outcome of three different prophylactic regimens in children with severe haemophilia A (primary) and which parameters caused variability in HJHS (secondary). METHODS: International retrospective observational multi-centre study comparing routine HJHS in 127 children with severe haemophilia A born from 1995 to 2009, from London, Stockholm and Utrecht centres. Patient and treatment data were collected from the European Paediatric Network for Haemophilia Management registry and patient files. The independent effects of regimens, physiotherapists, age and inhibitor status on HJHS were explored, using multivariable regression analysis. RESULTS: Prophylaxis varied across participating centres, with differences in initial frequency of infusions (1× per week vs. 3× per week), age at reaching infusions ≥3× per week, and dose kg(-1) week(-1) at HJHS assessment. Evaluation at median age of 11 years showed an illogical association of HJHS with treatment regimen: the least intensive regimen had the lowest HJHS. The HJHS increased with age and history of inhibitor, as expected (internal validity). But the comparison of prophylactic regimens was obscured by systematic differences in assessment between physiotherapists, both within and between centres. CONCLUSION: Inter-physiotherapist discrepancies in routine HJHS hamper comparison of scores between treatment regimens. For multi-centre research, additional inter-observer standardization for HJHS scoring is needed.

How to achieve full prophylaxis in young boys with severe haemophilia A: different regimens and their effect on early bleeding and venous access.

To facilitate early prophylaxis, step-up regimens starting prophylaxis with infusions 1× week(-1) were introduced. Choice of initial regimen may affect outcome. This study aims to classify initial prophylactic regimens and compare them on short-term outcome. From the 'European Paediatric Network for Haemophilia Management' (PedNet) registry, patients with severe haemophilia A without inhibitors, born 2000-2012, receiving prophylaxis were included. Treatment centres were classified according to the initial frequency of prophylactic infusions and the age at reaching infusions ≥3× week(-1) . Bleeding, and central venous access device (CVAD) use were compared at age 4 years. In 21 centres with 363 patients, three regimens were identified: (i) start prophylaxis with ≥3× week(-1) infusions before age three (full: 19% of centres, 18% of patients); (ii) start 1-2× week(-1) , increasing frequency as soon as possible (asap), reaching ≥3× week(-1) before age three (43% of centres, 36% of patients); (iii) start 1-2× week(-1) , increasing frequency according to bleeding (phenotype), reaching ≥3× week(-1) after age three (38% of centres, 46% of patients). Prophylaxis was started at median 1.2 years on the full and asap regimen vs 1.8 years on the phenotype regimen. Complete prevention of joint bleeds was most effective on the full regimen (32% full vs. 27% asap and 8% phenotype), though at the cost of using most CVADs (88% full vs. 34% asap and 22% phenotype). The three prophylaxis regimens identified had different effects on early bleeding and CVAD use. This classification provides the first step towards establishing the optimum prophylactic regimen.

Micro- and nano-hydroxyapatite as active reinforcement for soft biocomposites.

Pectin-based biocomposite hydrogels were produced by internal gelation, using different hydroxyapatite (HA) powders from commercial source or synthesized by the wet chemical method. HA possesses the double functionality of cross-linking agent and inorganic reinforcement. The mineralogical composition, grain size, specific surface area and microstructure of the hydroxyapatite powders are shown to strongly influence the properties of the biocomposites. Specifically, the grain size and specific surface area of the HA powders are strictly correlated to the gelling time and rheological properties of the hydrogels at room temperature. Pectin pH is also significant for the formation of ionic cross-links and therefore for the hydrogels stability at higher temperatures. The obtained results point out that micrometric-size hydroxyapatite can be proposed for applications which require rapid gelling kinetics and improved mechanical properties; conversely the nanometric hydroxyapatite synthesized in the present work seems the best choice to obtain homogeneous hydrogels with more easily controlled gelling kinetics.

External and internal gelation of pectin solutions: microscopic dynamics versus macroscopic rheology.

Pectin is a natural biopolymer that forms, in the presence of divalent cations, ionic-bound gels typifying a large class of biological gels stabilized by non-covalent cross-links. We investigate and compare the kinetics of formation and aging of pectin gels obtained either through external gelation via perfusion of free Ca(2+) ions, or by internal gelation due to the supply of the same ions from the dissolution of CaCO3 nanoparticles. The microscopic dynamics obtained with photon correlation imaging, a novel optical technique that allows obtaining the microscopic dynamics of the sample while retaining the spatial resolution of imaging techniques, is contrasted with macroscopic rheological measurements at constant strain. Pectin gelation is found to display peculiar two-stage kinetics, highlighted by non-monotonic growth in time of both microscopic correlations and gel mechanical strength. These results are compared to those found for alginate, another biopolymer extensively used in food formulation.

Similar bleeding phenotype in young children with haemophilia A or B: a cohort study.

The bleeding phenotype has been suggested to differ between haemophilia A and B. More knowledge on the bleeding phenotype at initiation of treatment is important to optimize patient care. The aim of this study was to investigate the severity of the bleeding phenotype and the variation in bleeding in children with severe or moderate haemophilia A and B. Consecutive, previously untreated patients with severe or moderate haemophilia A and B (factor VIII or IX activity <0.01 or 0.01-0.05 IU mL(-1) respectively) born between January 1st 2000 and January 1st 2010 were included. Primary outcome was severity of bleeding tendency. Secondary outcome was variation in bleeding pattern. A total of 582 patients with severe haemophilia A and 76 with severe haemophilia B did not differ in age at first exposure to clotting factor (0.81 vs. 0.88 years, P = 0.20), age at first bleed (0.82 vs. 0.88 years, P = 0.36), and age at first joint bleed (1.18 vs. 1.20 years, P = 0.59). Patients with moderate haemophilia were older compared to patients with severe haemophilia. In patients with moderate haemophilia there were no clear differences between haemophilia A and B. Severity and variation in bleeding phenotype are similar during the early stage of treatment in patients with severe and moderate haemophilia A and B respectively. The findings imply that children with haemophilia B should be observed and treated as vigilantly as those with haemophilia A.

Third Åland islands conference on von Willebrand disease, 26-28 September 2012: meeting report.

The first meeting of international specialists in the field of von Willebrand disease (VWD) was held in the Åland islands in 1998 where Erik von Willebrand had first observed a bleeding disorder in some members of a family from Föglö and a summary of the meeting was published in 1999. The second meeting was held in 2010 and a report of the meeting was published in 2012. Topics covered included progress in understanding of VWD over the last 50 years; multimers; classification of VWD; pharmacokinetics and laboratory assays; genetics; treating the paediatric patient; prophylaxis; geriatrics; gene therapy and treatment guidelines. This third meeting held over 3 days covered the structure and function of von Willebrand factor (VWF); type 1 VWD, the most common form of the disease; a lifespan of pharmacokinetics in VWD; detecting inhibitors in VWD patients; and special challenges in understanding and treating the female VWD patient.

von Willebrand's disease: a report from a meeting in the Åland islands.

von Willebrand's disease (VWD) is probably the most common bleeding disorder, with some studies indicating that up to 1% of the population may have the condition. Over recent years interest in VWD has fallen compared to that of haemophilia, partly the result of focus on blood-borne diseases such as HIV and hepatitis. Now the time has come to revisit VWD, and in view of this some 60 international physicians with clinical and scientific interest in VWD met over 4 days in 2010 in the Åland islands to discuss state-of-the-art issues in the disease. The Åland islands are where Erik von Willebrand had first observed a bleeding disorder in a number of members of a family from Föglö, and 2010 was also the 140th anniversary of his birth. This report summarizes the main papers presented at the symposium; topics ranged from genetics and biochemistry through to classification of VWD, pharmacokinetics and laboratory assays used in the diagnosis of the disease, inhibitors, treatment guidelines in different age groups including the elderly who often have comorbid conditions that present challenges, and prophylaxis. Other topics included managing surgeries in patients with VWD and the role of FVIII in VWF replacement, a controversial subject.

Advances in biomedical applications of pectin gels.

Pectin, due to its simple and cytocompatible gelling mechanism, has been recently exploited for different biomedical applications including drug delivery, gene delivery, wound healing and tissue engineering. Recent studies involving pectin for the biomedical field are reviewed, with the aim to capture the state of art on current research about pectin gels for biomedical applications, moving outside the traditional fields of application such as the food industry or pharmaceutics. Pectin structure, sources and extraction procedures have been discussed focussing on the properties of the polysaccharide that can be tuned to optimize the gels for a desired application and possess a fundamental role in application of pectin in the biomedical field.

Treatment of haemophilia A and B and von Willebrand's disease: summary and conclusions of a systematic review as part of a Swedish health-technology assessment.

In an ongoing health-technology assessment of haemophilia treatment in Sweden, performed by the governmental agency Dental and Pharmaceutical Benefits Agency (TLV; tandvårds-och lakemedelsförmånsverket), the Swedish Council on Health Technology Assessment (SBU; statens beredning för medicinsk utvardering) was called upon to evaluate treatment of haemophilia A and B and von Willebrand's disease (VWD) with clotting factor concentrates. To evaluate the following questions: What are the short-term and long-term effects of different treatment strategies? What methods are available to treat haemophilia patients that have developed inhibitors against factor concentrates? Based on the questions addressed by the project, a systematic database search was conducted in PubMed, NHSEED, Cochrane Library, EMBASE and other relevant databases. The literature search covered all studies in the field published from 1985 up to the spring of 2010. In most instances, the scientific evidence is insufficient for the questions raised in the review. Concentrates of coagulation factors have good haemostatic effects on acute bleeding and surgical intervention in haemophilia A and B and VWD, but conclusions cannot be drawn about possible differences in the effects of different dosing strategies for acute bleeding and surgery. Prophylaxis initiated at a young age can prevent future joint damage in persons with haemophilia. The available treatment options for inhibitors have been insufficiently assessed. The economic consequences of various treatment regimens have been insufficiently analysed. Introduction of national and international registries is important.

Joint health and functional ability in children with haemophilia who receive intensive replacement therapy.

Joint physical examination is an important outcome in haemophilia; however its relationship with functional ability is not well established in children with intensive replacement therapy. Boys aged 4-16 years were recruited from two European and three North American treatment centres. Joint physical structure and function was measured with the Haemophilia Joint Health Score (HJHS) while functional ability was measured with the revised Childhood Health Assessment Questionnaire (CHAQ38. Two haemophilia-specific domains were created by selecting items of the CHAQ38 that cover haemophilia-specific problems. Associations between CHAQ, HJHS, cumulative number of haemarthroses and age were assessed. A total of 226 subjects - mean 10.8 years old (SD 3.8) - participated; the majority (68%) had severe haemophilia. Most severe patients (91%) were on prophylactic treatment. Lifetime number of haemarthroses [median=5; interquartile range (IQR)=1-12] and total HJHS (median = 5; IQR=1-12) correlated strongly (ρ = 0.51). Total HJHS did not correlate with age and only weakly (ρ=-0.19) with functional ability scores (median=0; IQR=-0.06-0). Overall, haemarthroses were reported most frequently in the ankles. Detailed analysis of ankle joint health scores revealed moderate associations (ρ=0.3-0.5) of strength, gait and atrophy with lower extremity tasks (e.g. stair climbing). In this population, HJHS summating six joints did not perform as well as individual joint scores, however, certain elements of ankle impairment, specifically muscle strength, atrophy and gait associated significantly with functional loss in lower extremity activities. Mild abnormalities in ankle assessment by HJHS may lead to functional loss. Therefore, ankle joints may warrant special attention in the follow up of these children.

Pectin-based injectable biomaterials for bone tissue engineering.

A variety of natural polymers and proteins are considered to be 3D cell culture structures able to mimic the extracellular matrix (ECM) to promote bone tissue regeneration. Pectin, a natural polysaccharide extracted from the plant cell walls and having a chemical structure similar to alginate, provides interesting properties as artificial ECM. In this work, for the first time, pectin, modified with an RGD-containing oligopeptide or not, is used as an ECM alternative to immobilize cells for bone tissue regeneration. The viability, metabolic activity, morphology, and osteogenic differentiation of immobilized MC3T3-E1 preosteoblats demonstrate the potential of this polysaccharide to keep immobilized cells viable and differentiating. Preosteoblasts immobilized in both types of pectin microspheres maintained a constant viability up to 29 days and were able to differentiate. The grafting of the RGD peptide on pectin backbone induced improved cell adhesion and proliferation within the microspheres. Furthermore, not only did cells grow inside but also they were able to spread out from the microspheres and to organize themselves in 3D structures producing a mineralized extracellular matrix. These promising results suggest that pectin can be proposed as an injectable cell vehicle for bone tissue regeneration.

Structural properties of polysaccharide-based microcapsules for soft tissue regeneration.

Autologous and eterologous cell encapsulation has been extensively studied for clinical application in functional organs substitution, recombinant cell transplantation in gene therapy or in muscle and cartilage regeneration to treat degenerative pathologies. In this work, calcium alginate, calcium alginate/chitosan, calcium alginate/gelatin and pectin/chitosan microcapsules were prepared to be used as innovative injectable scaffolds for soft issue regeneration by a simple extrusion method from aqueous solutions. Prepared microcapsules had spherical morphology, whereas their size was deeply influenced by the polymeric composition. When incubated in a physiological-like environment up to 30 days, they underwent an initial swelling, followed by weight loss at different rates, depending on the microcapsules formulation. The encapsulation of mouse myoblast cells (C2C12 cell line) was obtained in calcium alginate, calcium alginate/chitosan, calcium alginate/gelatin microcapsules. Cells were alive throughout the encapsulation procedure, and were recovered by a mechanical rupture of the microcapsules. After 7 days, fractured microcapsules led cells to migrate gradually out.

Haemophilia care in adolescents--compliance and lifestyle issues.

Adolescence is characterized by simultaneous physical, psychological, social and sexual changes that compound the challenges faced by parents, health care providers and adolescent haemophilia patients themselves. Compliance with prophylactic factor replacement therapy frequently declines when patients pass from childhood to adolescence. Familiarity with long-term joint damage is lacking among the current generation of children who have grown up with prophylactic treatment and the tendency of teenagers to focus primarily on short-term goals increases the likelihood that regular prophylactic replacement therapy receives low priority. Most adolescents continue prophylactic treatment prior to physical or social activities because short-term goals are more likely to be perceived as relevant. The most important factor that influences compliance is support from parents, peers and caregivers, who provide encouragement and support active participation in health care management. During adolescence, personalized treatment strategies that suit the patient and his lifestyle are essential to ensure optimal outcomes. Physical activity is important for all adolescents and can contribute to better coordination, endurance, flexibility and strength. Physical training also contributes to healthier joints and reduces the risk of bleeding episodes in teenagers with haemophilia; however, the selection of an appropriate sport that minimizes the risk of injury and matches the patient's skill and needs is important. Children with haemophilia may have disease-related functional deficits and often exhibit subclinical findings in the joints; therefore an orthopaedic examination, fitness check and motion analysis may assist in guiding preventive physiotherapy and the choice of sport.

Clinical safety surveillance study of the safety and efficacy of long-term home treatment with ReFacto utilizing a computer-aided diary: a Nordic multicentre study.

A Nordic multicentre, open-label, non-interventional postmarketing surveillance study was carried out during a period of 24 months evaluating safety and efficacy of ReFacto as prophylactic or on-demand replacement therapy in patients with haemophilia A treated by self-medication. Fifty-seven patients were enrolled and studied for safety; efficacy was evaluated in 39 patients who received ReFacto for 24 months and recorded sufficient diary data on a hand-held computer. The compliance of using the device was good in small children, variable in adults and poor in teenagers. The fact that the overall compliance was low constituted a limitation of the number of patients with reliable diary data. Overall safety was rated as excellent or good by the clinicians for all patients at all visits and overall efficacy at 24 months evaluated to be excellent (74%) or good (26%). It was noticed that >/=50% of patients/parents reported no absences from school or work owing to bleeding episodes during the study period. Among patients on regular prophylaxis, 6 of the 30 patients (20%) receiving ReFacto experienced no bleeding episodes. A median of four bleeding episodes occurred during the 24-month study period, and 93% of the episodes were resolved with

Identifying and overcoming barriers to prophylaxis in the management of haemophilia.

Haemophilia is often characterized by acute haemarthrosis and synovitis arising from spontaneous bleeding episodes, particularly in the muscles and joints of the elbows, knees and ankles. Current treatment for patients with severe haemophilia involves coagulation factor concentrate (CFC) replacement therapy given on demand at the time of bleeding or through long-term prophylaxis aimed at preventing future bleeds and joint disease. Although prophylaxis has many advantages over on-demand therapy (particularly if started before age 2 and prior to any signs of joint disease), its practice varies widely even among developed countries because of several barriers. Such barriers include CFC costs and availability; patient perceptions, lifestyles and bleeding patterns; difficulties and complications arising from the use of intravenous access devices (IVADs); the development of inhibitors; and the lack of randomized clinical trials. These barriers can be overcome by tailoring treatment regimens according to individual patient bleeding patterns and CFC pharmacokinetic profiles, using IVADs selectively and judiciously, helping patients maintain normal weight and physical exercise and providing the families of patients with continuous support from healthcare providers.

Hemorrhagic symptoms and bleeding risk in obligatory carriers of type 3 von Willebrand disease: an international, multicenter study.

OBJECTIVES: We undertook an international, multicenter study to describe the clinical picture and to estimate the bleeding risk in a group of obligatory carriers of type 3 von Willebrand disease (VWD). PATIENTS AND METHODS: Obligatory carriers (OC) of type 3 VWD were identified by the presence of offspring with type 3 VWD or by being an offspring of a type 3 patient. Normal controls were age- and sex-matched with the obligatory carriers. A physician-administered standardized questionnaire was used to evaluate hemorrhagic symptoms at presentation. A score system ranging from 0 (no symptom) to 3 (hospitalization, replacement therapy, blood transfusion) was used to quantitate bleeding manifestations. Odds ratios were computed for each symptom. RESULTS: Ten centers participated to the study, enrolling a total of 35 type 3 VWD families, with 70 OC. A total of 215 normal controls and 42 OC for type 1 VWD were also included. About 40% of type 3 OC had at least one bleeding symptom compared to 23% of normal controls and 81.8% of type 1 OC (P < 0.0001 by chi-squared test), showing that type 3 OC clearly represent a distinct population from type 1 OC. The clinical situations associated with an increase of bleeding risk in type 3 OC were epistaxis [odds ratio 3.6; 90% confidence intervals (CI) 1.84-21.5], cutaneous bleeding (odds ratio 5.5; 90% CI 2.5-14.1) and postsurgical bleeding (odds ratio 16.3; 90% CI 4.5-59). The severity of bleeding score correlated with the degree of factor (F) VIII reduction in plasma. CONCLUSIONS: OC for type 3 VWD represent a distinctive population from type 1 OC. These patients, however, present with more frequent bleeding symptoms in comparison to normal controls, especially in case of significantly low FVIII. Desmopressin and/or tranexamic acid might be useful to prevent or treat bleeding in these cases.