Biosensing at Drop Scale Assisted by Electrowetting at Moderate Frequency

AbstractThis paper addresses surface ageing of a functionalized substrate in digital microfluidics. A model system based on bovine serum albumin (BSA)-laden sessile drop is selected. AC ElectroWetting-On-Dielectrics (EWOD), increasingly used in digital micro-systems for medical assays, is implemented in coplanar configuration. The small frequency re- quired by EWOD actuation (f =100 - 1000Hz) enables shape oscillations of the latter sessile drop and internal streaming. As demonstrated from dynamic contact angle (DCA) imaging, adsorption of BSA at the solid substrate is promoted un- der EWOD, as also shown from the time-dependent solid-liquid surface pressure. For an oscillating mode, n = 6, our numerical simulations show that corner eddies located near the oscillating contact line yield an efficient molecular re- newal. In this way, oscillating EWOD could be used purposely as a tool to speed up label-free detection from DCA measurements

Use of cell-free collagen type I matrix implants for the treatment of small cartilage defects in the knee: clinical and magnetic resonance imaging evaluation

Abstract Purpose Articular cartilage defects of the knee are a common condition for which several repair techniques have been described. The aim of the present study was to assess medium-term results of a one-step procedure using a cell-free collagen type I matrix. Methods Fifteen patients with articular cartilage defects of the knee were treated with an 11-mm-diameter cell-free collagen type 1 matrix implant. The matrices were implanted in a press-fit manner into the defect after careful debridement down to the subchondral bone but without penetration of this margin. Follow-up examinations were carried out at 6 weeks, 6 months, and at 12, 24, 36, and 48 months after implantation. Clinical assessment included the visual analogue scale (VAS), the Tegner activity scale, and the International Knee Documentation Committee (IKDC) score. Radiological assessment for graft attachment and tissue regeneration was performed using the magnetic observation of cartilage repair tissue (MOCART) score. Results A total of 15 patients (males: n = 6 and females: n = 9) with a mean age of 26.4 years (range 19-40) were treated. The mean VAS improved significantly when compared to the preoperative values (P \ 0.05). Six weeks after implantation, IKDC values were slightly lower than the preoperative values (n.s.), but increased significantly at final follow-up (P \ 0.05). At 24 months, there were no significant differences in the median Tegner score between the post-operative values and the preoperative values (n.s.). However, after 36 months, a significant improvement was noted that lasted at least up to 48 months (P \ 0.05). The MOCART score improved consistently up to 4 years after implantation, with significant improvements already observed after 12 months (P \ 0.05). No correlation between the clinical scores and the MOCART score could be perceived. Conclusion The present study showed that the use of cellfree collagen type I matrix implants led to a significant and durable improvement in all the clinical and imaging scores investigated 4 years after implantation. Level of evidence IV

Separable functions of wingless in distal and ventral patterning of the Tribolium leg

The gene wingless (wg) in Drosophila is an important factor in leg development. During embryonic development wg is involved in the allocation of the limb primordia. During imaginal disk development wg is involved in distal development and it has a separate role in ventral development. The expression pattern of wg is highly conserved in all arthropods (comprising data from insects, myriapods, crustaceans, and chelicerates), suggesting that its function in leg development is also conserved. However, recent work in other insects (e.g. the milkweed bug Oncopeltus fasciatus) argued against a role of wg in leg development. We have studied the role of wg in leg development of the flour beetle Tribolium castaneum. Using stage-specific staggered embryonic RNAi in wild-type and transgenic EGFP expressing enhancer trap lines we are able to demonstrate separable functions of Tribolium wg in distal and in ventral leg development. The distal role affects all podomeres distal to the coxa, whereas the ventral role is restricted to cells along the ventral midline of the legs. In addition, severe leg defects after injection into early embryonic stages are evidence that wg is also involved in proximal development and limb allocation in Tribolium. Our data suggest that the roles of wg in leg development are highly conserved in the holometabolous insects. Further studies will reveal the degree of conservation in other arthropod groups

Electrospun PLLA Nanofiber Scaffolds and Their Use in Combination with BMP-2 for Reconstruction of Bone Defects

Introduction Adequate migration and differentiation of mesenchymal stem cells is essential for regeneration of large bone defects. To achieve this, modern graft materials are becoming increasingly important. Among them, electrospun nanofiber scaffolds are a promising approach, because of their high physical porosity and potential to mimic the extracellular matrix (ECM). Materials and Methods The objective of the present study was to examine the impact of electrospun PLLA nanofiber scaffolds on bone formation in vivo, using a critical size rat calvarial defect model. In addition we analyzed whether direct incorporation of bone morphogenetic protein 2 (BMP-2) into nanofibers could enhance the osteoinductivity of the scaffolds. Two critical size calvarial defects (5 mm) were created in the parietal bones of adult male Sprague-Dawley rats. Defects were either (1) left unfilled, or treated with (2) bovine spongiosa, (3) PLLA scaffolds alone or (4) PLLA/BMP-2 scaffolds. Cranial CT-scans were taken at fixed intervals in vivo. Specimens obtained after euthanasia were processed for histology, histomorphometry and immunostaining (Osteocalcin, BMP-2 and Smad5). Results PLLA scaffolds were well colonized with cells after implantation, but only showed marginal ossification. PLLA/BMP-2 scaffolds showed much better bone regeneration and several ossification foci were observed throughout the defect. PLLA/BMP-2 scaffolds also stimulated significantly faster bone regeneration during the first eight weeks compared to bovine spongiosa. However, no significant differences between these two scaffolds could be observed after twelve weeks. Expression of osteogenic marker proteins in PLLA/BMP-2 scaffolds continuously increased throughout the observation period. After twelve weeks osteocalcin, BMP-2 and Smad5 were all significantly higher in the PLLA/BMP-2 group than in all other groups. Conclusion Electrospun PLLA nanofibers facilitate colonization of bone defects, while their use in combination with BMP-2 also increases bone regeneration in vivo and thus combines osteoconductivity of the scaffold with the ability to maintain an adequate osteogenic stimulus

Expression, localization and polymorphisms of the nuclear receptor PXR in Barrett's esophagus and esophageal adenocarcinoma

Background: The continuous exposure of esophageal epithelium to refluxate may induce ectopic expression of bile-responsive genes and contribute to the development of Barrett's esophagus (BE) and esophageal adenocarcinoma. In normal physiology of the gut and liver, the nuclear receptor Pregnane × Receptor (PXR) is an important factor in the detoxification of xenobiotics and bile acid homeostasis. This study aimed to investigate the expression and genetic variation of PXR in reflux esophagitis (RE), Barrett's esophagus (BE) and esophageal adenocarcinoma.Methods: PXR mRNA levels and protein expression were determined in biopsies from patients with adenocarcinoma, BE, or RE, and healthy controls. Esophageal cell lines were stimulated with lithocholic acid and rifampicin. PXR polymorphisms 25385C/T, 7635A/G, and 8055C/T were genotyped in 249 BE patients, 233 RE patients, and 201 controls matched for age and gender.Results: PXR mRNA levels were significantly higher in adenocarcinoma tissue and columnar Barrett's epithelium, compared to squamous epithelium of these BE patients (P < 0.001), and RE patients (P = 0.003). Immunohistochemical staining of PXR showed predominantly cytoplasmic expression in BE tissue, whereas nuclear expression was found in adenocarcinoma tissue. In cell lines, stimulation with lithocholic acid did not increase PXR mRNA levels, but did induce nuclear translocation of PXR protein. Genotyping of the PXR 7635A/G polymorphism revealed that the G allele was significantly more prevalent in BE than in RE or controls (P = 0.037).Conclusions: PXR expresses in BE and adenocarcinoma tissue, and showed nuclear localization in adenocarcinoma tissue. Upon stimulation with lithocholic acid, PXR translocates to the nuclei of OE19 adenocarcinoma cells. Together with the observed association of a PXR polymorphism and BE, this data implies that PXR may have a function in prediction and treatment of esophageal disease

Electromouillage bi-fréquence et interférométrie : évaporation et vieillissement biochimique à l’échelle d’une goutte

This PhD research is thought of as a first step towards label freedigital biosensing. A novel transduction mechanism is developed, based on thegeneration of capillary waves along a drop surface induced by dual-frequencyelectrowetting. Acapillary resonance is measured by interferometry at the movingdrop apex. Aproof of concept of this electro-optical methodology for furtherintegration in a digital microsystem is given with characterization of drop evaporationkinetics. A second part focuses on modeling and numerical calculationsof the transport of surface active biomolecules in the drop. The model includesdiffusion, ad-/desorption phenomena together with a kinematic condition of avariable drop radius (evaporation) as well as a finite molecular amount of surfaceactive biomolecules. A dimensionless number is suggested to take into accountfinite size effects. Drop steady streaming due to oscillating electrowetting isfinally taken into account. Low- and high-frequency electrowetting is appliedto a drop laden with bovine serum albumin. By jointly using interferometry andimaging, enhancement of surface ageing is made evident under electrowetting.La recherche développée durant cette thèse constitue la première étape de développement d’une nouvelle méthodologie de détection sans marquage à l’échelle d’une goutte : un nouveau mécanisme de transduction est développé, basé sur l’actuation par électromouillage bi-fréquence d’un réseau d’ondes capillaires à la surface d’une goutte. La résonance capillaire de la goutte est étudiée par détection interférométrique de son apex en mouvement.La faisabilité de cette méthodologie est prouvée en caractérisant la cinétique d’évaporation de la goutte par glissement spectral des ondes capillaires.La deuxième partie de la thèse porte sur la modélisation du transport de biomolécules tensioactives dans une goutte : le transport diffusif, les processus d’ad/désorption aux surfaces, la condition cinématique d’un rayon variable(évaporation) ainsi qu’une quantité molaire finie de biomolécules sont pris en compte. Un nouveau nombre adimensionnel est proposé pour rendre compte de la taille finie d’une goutte. Puis le transport chimique associé au courant de dérive induit par les ondes capillaires (excitées par électromouillage oscillatoire)est pris en compte à l’aide de simulations numériques. Finalement,l’électromouillage haute et basse fréquence est appliqué au cas d’une goutte enrichie par des molécules d’albumine de sérum bovin. En utilisant conjointement l’interférométrie optique et l’imagerie, et en considérant la loi de Lippmann-Young, le vieillissement biochimique de la surface solide mouillée par la goutte est détecté

Mass transfer enhancement and surface functionalization in digital microfluidics using AC electrowetting: the smaller, the better

International audienceThis paper addresses modelling and computation of stirring and chemical transport at drop scale due to shape oscillations of small amplitude induced by coplanar AC electrowetting on dielectrics (EWOD). An axisymmetric flow inside a drop deformed by standing capillary waves is promoted by coplanar EWOD in the oscillating regime ( 10-500 Hz). This can be seen as a new stirring/mixing mechanism at microscale provided that a deformable fluid interface is available. The latter requirement is fully satisfied in biomedical applications of digital microfluidics where droplets of volume similar to 1 mu L are commonly handled. A steady streaming model at large Strouhal number is taken into account in order to investigate time-dependent chemical transport in a sessile drop under oscillating EWOD. Numerical calculations based on finite element method are performed to predict the transport of a biochemical solute with possible affinity for drop surface and supporting solid surface as well. A dedicated weak form is also derived to take into account diffusional transport along the liquid surface. Mass transfer enhancement is investigated by considering the variance of the chemical concentration in the transient regime. Special attention is given to investigate mass transfers and surface ageing by considering the respective roles of the Thiele number, the Damkohler number, the Peclet and Surface Peclet numbers