Superhydrophobic platforms for the combinatorial analysis of biomaterials-cells interactions using arrays of 3D scaffolds with distinct mechanical and morphological properties

High-throughput studies of cells mechanotransduction are usually performed using 2D biomaterials. However, cells-extracellular matrix interactions in the body occur in 3D environment. By using cells entrapped in hydrogels, it is not easy to isolate the mechanical effect from the chemical cues of biomaterials. We used a cytocompatible and non-expensive platform based in the patterning of wettable spots in superhydrophobic surfaces to deposit porous biomaterials in these regions. Freeze-dried alginate/chitosan scaffolds were used to create an array of mechanical properties and porosities. Adaptation of dynamic mechanic analysis equipment allowed performing on-chip single scaffold analysis. Micro-computed tomography allowed acquir- ing data for whole chips simultaneously. Results were validated using individual scaffolds and single-formulation chips. A sub-array with combined modulus/porosity properties was selected. Fibronectin (Fn) in different concentration was adsorbed in the scaffolds, and fibroblasts and osteoblast-like cells were seeded. The independent study of variables influence in cell response was performed by image-based methods. In the absence of Fn fibroblasts did not respond to mechani- cal properties in the chip range. Osteoblast-like cells showed higher cell adhesion in stiffer substrates. The adsorption of Fn was studied qualita- tively by image methods. Results related with Fn amount and scaffolds’ mechanical properties were analyzed for each cell type.</p

Characterization of chitosan and polycaprolactone membranes designed for wound repair application

Polycaprolactone (PCL) and chitosan (Ch) are nontoxic, biocompatible, and biodegradable polymers of vast interest for wound repair. The aim of this work was to prepare Ch/PCL membranes in different proportions (90:10 and 80:20 w/w) in the presence and absence of the surfactant Pluronic F68 (PF68). The membranes were evaluated regarding morphology, thermal behavior, and viscoelastic properties. Sample swelling and degradation in phosphate-buffered saline (PBS), simulated body fluid (SBF), and fetal bovine serum (FBS) were determined by differential scanning calorimetry (DSC) and dynamical mechanical analysis (DMA), while cell toxicity to L929 and Vero fibroblasts was evaluated using the MTT reduction assay and cell proliferation, by DNA quantification and confocal laser microscopy. After 60 days in SBF, marked Ch matrix loss and advanced degradation of PCL particles were noticed by scanning electron microscopy (SEM). No significant differences in melting temperature (Tm) and enthalpy (DHm) were detected by DSC. However, the surfactant increased the DHm. After 30 days, the membranes obtained in the presence of PF68 had absorbed more blood serum and were more degraded after exposure to simulated blood fluid for 30 days. All membranes had low cytotoxicity, and higher cell proliferation was noticed for samples obtained in the presence of the surfactant. In conclusion, the Ch/PCL membranes showed satisfactory degradability and biocompatibility, which enhances their potential for application in wound repair.The authors thank the PhD student Sofia Caridade (3B's Research Group-Universidade do Minho, Portugal) for her assistance in the DMA analyses. The financial support provided by the Conselho Nacional de Desenvolvimento Cientifico e Tecnologico (CNPq-150984/2009-0) in Brazil is gratefully acknowledged for this work

TRY plant trait database – enhanced coverage and open access

Plant traits - the morphological, anatomical, physiological, biochemical and phenological characteristics of plants - determine how plants respond to environmental factors, affect other trophic levels, and influence ecosystem properties and their benefits and detriments to people. Plant trait data thus represent the basis for a vast area of research spanning from evolutionary biology, community and functional ecology, to biodiversity conservation, ecosystem and landscape management, restoration, biogeography and earth system modelling. Since its foundation in 2007, the TRY database of plant traits has grown continuously. It now provides unprecedented data coverage under an open access data policy and is the main plant trait database used by the research community worldwide. Increasingly, the TRY database also supports new frontiers of trait‐based plant research, including the identification of data gaps and the subsequent mobilization or measurement of new data. To support this development, in this article we evaluate the extent of the trait data compiled in TRY and analyse emerging patterns of data coverage and representativeness. Best species coverage is achieved for categorical traits - almost complete coverage for ‘plant growth form’. However, most traits relevant for ecology and vegetation modelling are characterized by continuous intraspecific variation and trait–environmental relationships. These traits have to be measured on individual plants in their respective environment. Despite unprecedented data coverage, we observe a humbling lack of completeness and representativeness of these continuous traits in many aspects. We, therefore, conclude that reducing data gaps and biases in the TRY database remains a key challenge and requires a coordinated approach to data mobilization and trait measurements. This can only be achieved in collaboration with other initiatives

Peri-operative red blood cell transfusion in neonates and infants: NEonate and Children audiT of Anaesthesia pRactice IN Europe: A prospective European multicentre observational study

BACKGROUND: Little is known about current clinical practice concerning peri-operative red blood cell transfusion in neonates and small infants. Guidelines suggest transfusions based on haemoglobin thresholds ranging from 8.5 to 12 g dl-1, distinguishing between children from birth to day 7 (week 1), from day 8 to day 14 (week 2) or from day 15 (≥week 3) onwards. OBJECTIVE: To observe peri-operative red blood cell transfusion practice according to guidelines in relation to patient outcome. DESIGN: A multicentre observational study. SETTING: The NEonate-Children sTudy of Anaesthesia pRactice IN Europe (NECTARINE) trial recruited patients up to 60 weeks' postmenstrual age undergoing anaesthesia for surgical or diagnostic procedures from 165 centres in 31 European countries between March 2016 and January 2017. PATIENTS: The data included 5609 patients undergoing 6542 procedures. Inclusion criteria was a peri-operative red blood cell transfusion. MAIN OUTCOME MEASURES: The primary endpoint was the haemoglobin level triggering a transfusion for neonates in week 1, week 2 and week 3. Secondary endpoints were transfusion volumes, 'delta haemoglobin' (preprocedure - transfusion-triggering) and 30-day and 90-day morbidity and mortality. RESULTS: Peri-operative red blood cell transfusions were recorded during 447 procedures (6.9%). The median haemoglobin levels triggering a transfusion were 9.6 [IQR 8.7 to 10.9] g dl-1 for neonates in week 1, 9.6 [7.7 to 10.4] g dl-1 in week 2 and 8.0 [7.3 to 9.0] g dl-1 in week 3. The median transfusion volume was 17.1 [11.1 to 26.4] ml kg-1 with a median delta haemoglobin of 1.8 [0.0 to 3.6] g dl-1. Thirty-day morbidity was 47.8% with an overall mortality of 11.3%. CONCLUSIONS: Results indicate lower transfusion-triggering haemoglobin thresholds in clinical practice than suggested by current guidelines. The high morbidity and mortality of this NECTARINE sub-cohort calls for investigative action and evidence-based guidelines addressing peri-operative red blood cell transfusions strategies. TRIAL REGISTRATION: ClinicalTrials.gov, identifier: NCT02350348

Retrospective evaluation of whole exome and genome mutation calls in 746 cancer samples

Funder: NCI U24CA211006Abstract: The Cancer Genome Atlas (TCGA) and International Cancer Genome Consortium (ICGC) curated consensus somatic mutation calls using whole exome sequencing (WES) and whole genome sequencing (WGS), respectively. Here, as part of the ICGC/TCGA Pan-Cancer Analysis of Whole Genomes (PCAWG) Consortium, which aggregated whole genome sequencing data from 2,658 cancers across 38 tumour types, we compare WES and WGS side-by-side from 746 TCGA samples, finding that ~80% of mutations overlap in covered exonic regions. We estimate that low variant allele fraction (VAF < 15%) and clonal heterogeneity contribute up to 68% of private WGS mutations and 71% of private WES mutations. We observe that ~30% of private WGS mutations trace to mutations identified by a single variant caller in WES consensus efforts. WGS captures both ~50% more variation in exonic regions and un-observed mutations in loci with variable GC-content. Together, our analysis highlights technological divergences between two reproducible somatic variant detection efforts

Patterned superhydrophobic surfaces for the combinatorial assessment of 3D biomaterials-cells interactions

Cells-biomaterials interactions are dependent on a wide range of fac- tors, namely substrate mechanical properties. Usually in the study of mechanotransduction phenomena 2D models are used and surface modification of the biomaterials is performed to suppress the effect of materials chemistry. However, 3D structures provide a more accurate mimicry of the physiological media. In high-throughput analysis cell encapsulation is commonly used, although in different stiffness hydro- gels the maintenance of chemical features is difficult to achieve. Wetta- ble spots were patterned in superhydrophobic surfaces in order to deposit polymeric precursors and generate combinatorial porous scaf- folds. On-chip dynamic mechanical analysis was performed, as well as simultaneous quantification of porosity of several scaffolds. After select- ing groups of scaffolds with a wide range of mechanical and morpho- logical properties, a new array was created, consisting of these scaffolds with fibronectin adsorbed through the whole solid structure. Protein adsorption was studied by image analysis. The adhesion of two different cell types was studied. A tendency for an increased metabolic activity/cell number rate could be observed for pre-osteoblast cells in high stiffness scaffolds, while an opposite tendency was verified for fi- broblasts. In both cell types, mainly for pre-osteblasts, a tendency for increased metabolic activity and cell number in the scaffolds in the presence of fibronectin was observed

Combinatorial cell-porous scaffolds interactions study and scaffold physicochemical characterization in an innovative bioinspired high-throughput platform

Superhydrophobic surfaces patterned with size-controlled hydrophilic spots are proposed for the production of porous scaffolds arrays for combinatorial biomaterial-cells interaction studies. This approach allows for the open and easy access to scaffolds for further follow-ups aimed at highthroughput screening, when compared with the other scaffold array production techniques using commercial well plates or soft lithography-produced wells. Mechanical and morphological characterization along with cell study using chitosan/alginate mixtures with different concentrations of fibronectin were the proposed objectives

Combinatorial cell-3D biomaterials cytocompatibility screening for tissue engineering using bioinspired superhydrophobic substrates

We report on the development of a new array-based screening flat platform with the potential to be used as a high-throughput device based on biomimetic polymeric substrates for combinatorial cell/3D biomaterials screening assays in the context of tissue engineering. Polystyrene was used to produce superhydrophobic surfaces based on the so-called lotus effect. Arrays of hydrophilic regions could be patterned in such surfaces using UV/ozone radiation, generating devices onto which combinatorial hydrogel spots were deposited. The biological performance of encapsulated cells in hydrogels could be tested in an in vitro 3D environment assuming that each site was isolated from the others due to the high contrast of wettability between the patterned spots and the superhydrophobic surroundings. Three different polymers-chitosan, collagen and hyaluronic acid-were combined with alginate in different proportions in order to obtain combinatorial binary alginate-based polymeric arrays. The effect of the addition of gelatin to the binary structures was also tested. The gels were chemically analyzed by FTIR microscopic mapping. Cell culture results varied according to the hydrogel composition and encapsulated cell types (L929 fibroblast cells and MC3T3-E1 pre-osteoblast cells). Cell viability and number could be assessed by conventional methods, such as MTS reduction test and dsDNA quantification. Non-destructive image analysis was performed using cytoskeleton and nuclei staining agents and the results were consistent with the ones obtained by conventional sample-destructive techniques. Briefly, L929 cells showed higher number and viability for higher alginate-content and collagen-containing hydrogels, while MC3T3-E1 showed higher cell viability and cell number in lower alginate-content and chitosan containing hydrogels. The addition of gelatin did not influence significantly cell metabolic activity or cell number in any of the encapsulated cell types.Fundação para a Ciência e a Tecnologia (FCT) - SFRH/BD/71396/201

Superhydrophobic patterned chips for the combinatorial and rapid study of 3D biomaterials-cells interactions and protein delivery systems

The development of optimized products in the tissue engineering (TE) field is a time and resource consuming process due to the unpredictable influence of the combination of variables such as biomaterials, cells and soluble factors. As multiple combinations can be considered highthroughput (HT) methods were suggested as a way to master complexity in TE. Usually, HT systems test cell-2D biomaterials interactions and, more recently, cell-3D hydrogels interactions. Using a chip consisting of superhydrophobic surfaces patterned with wettable regions we tested cells-hydrogels interactions in three-dimensional environment [1]. The versatility of the chip allowed its use for the first time on-chip combinatorial study of 3D miniaturized porous scaffolds. Arrays of biomaterials were dispensed and processed in situ as porous scaffolds with distinct composition, surface characteristics, porosity/pore size and mechanical properties. Those characteristics were assessed by adapting microcomputed tomography equipment and a dynamic mechanical analyzer. The interactions between cell types of two distinct origins – osteoblast-like and fibroblasts - and the scaffolds modified with distinct amounts of fibronectin were studied by image-based methods and validated by comparison with conventional destructive methods. Physical and biological on-chip results were coherent with conventional measures, and conclusions about the most favorable media for the growth of both cell types were taken. Growth factors (GF) proved to play an important role in TE approaches, mainly for determining cell fate in applications containing stem cells. We developed a chip based on wettability contrast with torus-shaped hydrophilic transparent regions disposed in an array matrix. Concentrically to these wettable regions a superhydrophobic circle was maintained, so the hydrogels could be processed as protein-loaded spheres with minimum protein loss [2] and fixed with an indentation. A combinatorial system of BSA-FITC – a commonly used GF model – encapsulated in alginate hydrogels was designed. The protein release from the hydrogels could be studied by image analysis, avoiding manipulation and protein loss. The results were compared with conventional protein release tests and similar tendencies were observed. We believe that the proposed innovative uses for the superhydrophobic chip and their upgrade in future applications may constitute a promising breakthrough in integrated technologies for the rapid development of TE systems