Resealable Antithrombotic Artificial Vascular Graft Integrated with a Self-Healing Blood Flow Sensor.

Coronary artery bypass grafting is commonly used to treat cardiovascular diseases by replacing blocked blood vessels with autologous or artificial blood vessels. Nevertheless, the availability of autologous vessels in infants and the elderly and low long-term patency rate of grafts hinder extensive application of autologous vessels in clinical practice. The biological and mechanical properties of the resealable antithrombotic artificial vascular graft (RAAVG) fabricated herein, comprising a bioelectronic conduit based on a tough self-healing polymer (T-SHP) and a lubricious inner coating, match with the functions of autologous blood vessels. The self-healing and elastic properties of the T-SHP confer resistance against mechanical stimuli and promote conformal sealing of suturing regions, thereby preventing leakage (stable fixation under a strain of 50%). The inner layer of the RAAVG presents antibiofouling properties against blood cells and proteins, and antithrombotic properties, owing to its lubricious coating. Moreover, the blood-flow sensor fabricated using the T-SHP and carbon nanotubes is seamlessly integrated into the RAAVG via self-healing and allows highly sensitive monitoring of blood flow at low and high flow rates (10- and 100 mL min-1, respectively). Biocompatibility and feasibility of RAAVG as an artificial graft were demonstrated via ex vivo, and in vivo experiment using a rodent model. The use of RAAVGs to replace blocked blood vessels can improve the long-term patency rate of coronary artery bypass grafts.

Combining Image Restoration and Traction Force Microscopy to Study Extracellular Matrix-Dependent Keratin Filament Network Plasticity.

Keratin intermediate filaments are dynamic cytoskeletal components that are responsible for tuning the mechanical properties of epithelial tissues. Although it is known that keratin filaments (KFs) are able to sense and respond to changes in the physicochemical properties of the local niche, a direct correlation of the dynamic three-dimensional network structure at the single filament level with the microenvironment has not been possible. Using conventional approaches, we find that keratin flow rates are dependent on extracellular matrix (ECM) composition but are unable to resolve KF network organization at the single filament level in relation to force patterns. We therefore developed a novel method that combines a machine learning-based image restoration technique and traction force microscopy to decipher the fine details of KF network properties in living cells grown on defined ECM patterns. Our approach utilizes Content-Aware Image Restoration (CARE) to enhance the temporal resolution of confocal fluorescence microscopy by at least five fold while preserving the spatial resolution required for accurate extraction of KF network structure at the single KF/KF bundle level. The restored images are used to segment the KF network, allowing numerical analyses of its local properties. We show that these tools can be used to study the impact of ECM composition and local mechanical perturbations on KF network properties and corresponding traction force patterns in size-controlled keratinocyte assemblies. We were thus able to detect increased curvature but not length of KFs on laminin-322 versus fibronectin. Photoablation of single cells in microprinted circular quadruplets revealed surprisingly little but still significant changes in KF segment length and curvature that were paralleled by an overall reduction in traction forces without affecting global network orientation in the modified cell groups irrespective of the ECM coating. Single cell analyses furthermore revealed differential responses to the photoablation that were less pronounced on laminin-332 than on fibronectin. The obtained results illustrate the feasibility of combining multiple techniques for multimodal monitoring and thereby provide, for the first time, a direct comparison between the changes in KF network organization at the single filament level and local force distribution in defined paradigms.

Quantitative mapping of keratin networks in 3D.

Mechanobiology requires precise quantitative information on processes taking place in specific 3D microenvironments. Connecting the abundance of microscopical, molecular, biochemical, and cell mechanical data with defined topologies has turned out to be extremely difficult. Establishing such structural and functional 3D maps needed for biophysical modeling is a particular challenge for the cytoskeleton, which consists of long and interwoven filamentous polymers coordinating subcellular processes and interactions of cells with their environment. To date, useful tools are available for the segmentation and modeling of actin filaments and microtubules but comprehensive tools for the mapping of intermediate filament organization are still lacking. In this work, we describe a workflow to model and examine the complete 3D arrangement of the keratin intermediate filament cytoskeleton in canine, murine, and human epithelial cells both, in vitro and in vivo. Numerical models are derived from confocal airyscan high-resolution 3D imaging of fluorescence-tagged keratin filaments. They are interrogated and annotated at different length scales using different modes of visualization including immersive virtual reality. In this way, information is provided on network organization at the subcellular level including mesh arrangement, density and isotropic configuration as well as details on filament morphology such as bundling, curvature, and orientation. We show that the comparison of these parameters helps to identify, in quantitative terms, similarities and differences of keratin network organization in epithelial cell types defining subcellular domains, notably basal, apical, lateral, and perinuclear systems. The described approach and the presented data are pivotal for generating mechanobiological models that can be experimentally tested.

Regulation of keratin network dynamics by the mechanical properties of the environment in migrating cells.

Keratin intermediate filaments provide mechanical resilience for epithelia. They are nevertheless highly dynamic and turn over continuously, even in sessile keratinocytes. The aim of this study was to characterize and understand how the dynamic behavior of the keratin cytoskeleton is integrated in migrating cells. By imaging human primary keratinocytes producing fluorescent reporters and by using standardized image analysis we detect inward-directed keratin flow with highest rates in the cell periphery. The keratin flow correlates with speed and trajectory of migration. Changes in fibronectin-coating density and substrate stiffness induces concordant changes in migration speed and keratin flow. When keratinocytes are pseudo-confined on stripes, migration speed and keratin flow are reduced affecting the latter disproportionately. The regulation of keratin flow is linked to the regulation of actin flow. Local speed and direction of keratin and actin flow are very similar in migrating keratinocytes with keratin flow lagging behind actin flow. Conversely, reduced actin flow in areas of high keratin density indicates an inhibitory function of keratins on actin dynamics. Together, we propose that keratins enhance persistence of migration by directing actin dynamics and that the interplay of keratin and actin dynamics is modulated by matrix adhesions.

Keratin intermediate filaments: intermediaries of epithelial cell migration.

Migration of epithelial cells is fundamental to multiple developmental processes, epithelial tissue morphogenesis and maintenance, wound healing and metastasis. While migrating epithelial cells utilize the basic acto-myosin based machinery as do other non-epithelial cells, they are distinguished by their copious keratin intermediate filament (KF) cytoskeleton, which comprises differentially expressed members of two large multigene families and presents highly complex patterns of post-translational modification. We will discuss how the unique mechanophysical and biochemical properties conferred by the different keratin isotypes and their modifications serve as finely tunable modulators of epithelial cell migration. We will furthermore argue that KFs together with their associated desmosomal cell-cell junctions and hemidesmosomal cell-extracellular matrix (ECM) adhesions serve as important counterbalances to the contractile acto-myosin apparatus either allowing and optimizing directed cell migration or preventing it. The differential keratin expression in leaders and followers of collectively migrating epithelial cell sheets provides a compelling example of isotype-specific keratin functions. Taken together, we conclude that the expression levels and specific combination of keratins impinge on cell migration by conferring biomechanical properties on any given epithelial cell affecting cytoplasmic viscoelasticity and adhesion to neighboring cells and the ECM.

Hemidesmosomes and Focal Adhesions Treadmill as Separate but Linked Entities during Keratinocyte Migration.

Hemidesmosomes anchor the epidermal keratin filament cytoskeleton to the extracellular matrix. They are crucial for the mechanical integrity of skin. Their role in keratinocyte migration, however, remains unclear. Examining migrating primary human keratinocytes, we find that hemidesmosomes cluster as ordered arrays consisting of multiple chevrons that are flanked by actin-associated focal adhesions. These hemidesmosomal arrays with intercalated focal adhesions extend from the cell rear to the cell front. New hemidesmosomal chevrons form subsequent to focal adhesion assembly at the cell's leading front, whereas chevrons and associated focal adhesions disassemble at the cell rear in reverse order. The bulk of the hemidesmosome-focal adhesion composite, however, remains attached to the substratum during cell translocation. Similar hemidesmosome-focal adhesion patterns emerge on X-shaped fibronectin-coated micropatterns, during cell spreading and in leader cells during collective cell migration. We further find that hemidesmosomes and focal adhesions affect each other's distribution. We propose that both junctions are separate but linked entities, which treadmill coordinately to support efficient directed cell migration and cooperate to coordinate the dynamic interplay between the keratin and actin cytoskeleton.

Hierarchical Extended Bilateral Motion Estimation-Based Frame Rate Upconversion Using Learning-Based Linear Mapping.

We present a novel and effective learning-based frame rate upconversion (FRUC) scheme, using linear mapping. The proposed learning-based FRUC scheme consists of: 1) a new hierarchical extended bilateral motion estimation (HEBME) method; 2) a light-weight motion deblur (LWMD) method; and 3) a synthesis-based motion-compensated frame interpolation (S-MCFI) method. First, the HEBME method considerably enhances the accuracy of the motion estimation (ME), which can lead to a significant improvement of the FRUC performance. The proposed HEBME method consists of two ME pyramids with a three-layered hierarchy, where the motion vectors (MVs) are searched in a coarse-to-fine manner via each pyramid. The found MVs are further refined in an enhanced resolution of four times by jointly combining the MVs from the two pyramids. The HEBME method employs a new elaborate matching criterion for precise ME which effectively combines a bilateral absolute difference, an edge variance, pixel variances, and an MV difference among two consecutive blocks and its neighboring blocks. Second, the LWMD method uses the MVs found by the HEBME method and removes the small motion blurs in original frames via transformations by linear mapping. Third, the S-MCFI method finally generates interpolated frames by applying linear mapping kernels for the deblurred original frames. In consequence, our FRUC scheme is capable of precisely generating interpolated frames based on the HEBME for accurate ME, the S-MCFI for elaborate frame interpolation, and the LWMD for contrast enhancement. The experimental results show that our FRUC significantly outperforms the state-of-the-art non-deep learning-based schemes with an average of 1.42 dB higher in the peak signal-to-noise-ratio and shows comparable performance with the state-of-the-art deep learning-based scheme.

Droplet-based microfluidic system to form and separate multicellular spheroids using magnetic nanoparticles.

The importance of creating a three-dimensional (3-D) multicellular spheroid has recently been gaining attention due to the limitations of monolayer cell culture to precisely mimic in vivo structure and cellular interactions. Due to this emerging interest, researchers have utilized new tools, such as microfluidic devices, that allow high-throughput and precise size control to produce multicellular spheroids. We have developed a droplet-based microfluidic system that can encapsulate both cells and magnetic nanoparticles within alginate beads to mimic the function of a multicellular tumor spheroid. Cells were entrapped within the alginate beads along with magnetic nanoparticles, and the beads of a relatively uniform size (diameters of 85% of the beads were 170-190 µm) were formed in the oil phase. These beads were passed through parallel streamlines of oil and culture medium, where the beads were magnetically transferred into the medium phase from the oil phase using an external magnetic force. This microfluidic chip eliminates additional steps for collecting the spheroids from the oil phase and transferring them to culture medium. Ultimately, the overall spheroid formation process can be achieved on a single microchip.

Regulation of morphogenesis and neural differentiation of human mesenchymal stem cells using carbon nanotube sheets.

In order to successfully utilize stem cells for therapeutic applications in regenerative medicine, efficient differentiation into a specific cell lineage and guidance of axons in a desired direction is crucial. Here, we used aligned multi-walled carbon nanotube (MWCNT) sheets to differentiate human mesenchymal stem cells (hMSCs) into neural cells. Human MSCs present a preferential adhesion to aligned CNT sheets with longitudinal stretch parallel to the CNT orientation direction. Cell elongation was 2-fold higher than the control and most of the cells were aligned on CNT sheets within 5° from the CNT orientation direction. Furthermore, a significant, synergistic enhancement of neural differentiation was observed in hMSCs cultured on the CNT sheets. Axon outgrowth was also controlled using nanoscale patterning of CNTs. This CNT sheet provides a new cellular scaffold platform that can regulate morphogenesis and differentiation of stem cells, which could open up a new approach for tissue and stem cell regeneration.

Enhancement of neurite outgrowth in PC12 cells by iron oxide nanoparticles.

Despite the many potential therapeutic applications of iron oxide nanoparticle such as its use as an imaging and targeting tool, its biological effects have not yet been extensively characterized. Herein, we report that iron oxide nanoparticles taken up by PC12 cells can enhance neurite outgrowth. PC12 cells exposed to both iron oxide nanoparticles and nerve growth factor (NGF) synergistically increased the efficiency of neurite outgrowth in a dose-dependent manner. This may have resulted from the activation of cell adhesion molecules that are associated with cell-matrix interactions through iron. Immunoblotting assays also revealed that both neural specific marker protein and cell adhesion protein expression were upregulated by iron oxide nanoparticles compared with non-treated cells via activation of the mitogen-activated protein kinase (MAPK) signaling pathway. Our findings point to the possibility that iron oxide nanoparticles can affect cell-substrate interactions and regulate cell behaviors, which provides clinical insights into potential neurologic and therapeutic applications of iron oxide nanoparticles.