Complex hemolymph circulation patterns in grasshopper wings.

An insect's living systems-circulation, respiration, and a branching nervous system-extend from the body into the wing. Wing hemolymph circulation is critical for hydrating tissues and supplying nutrients to living systems such as sensory organs across the wing. Despite the critical role of hemolymph circulation in maintaining healthy wing function, wings are often considered "lifeless" cuticle, and flows remain largely unquantified. High-speed fluorescent microscopy and particle tracking of hemolymph in the wings and body of the grasshopper Schistocerca americana revealed dynamic flow in every vein of the fore- and hindwings. The global system forms a circuit, but local flow behavior is complex, exhibiting three distinct types: pulsatile, aperiodic, and "leaky" flow. Thoracic wing hearts pull hemolymph from the wing at slower frequencies than the dorsal vessel; however, the velocity of returning hemolymph (in the hindwing) is faster than in that of the dorsal vessel. To characterize the wing's internal flow mechanics, we mapped dimensionless flow parameters across the wings, revealing viscous flow regimes. Wings sustain ecologically important insect behaviors such as pollination and migration. Analysis of the wing circulatory system provides a template for future studies investigating the critical hemodynamics necessary to sustaining wing health and insect flight.

Investigation of macromolecular transport through tunable collagen hyaluronic acid matrices.

Therapeutic macromolecules possess properties such as size and electrostatic charge that will dictate their transport through subcutaneous (SC) tissue and ultimate bioavailability and efficacy. To improve therapeutic design, platforms that systematically measure the transport of macromolecules as a function of both drug and tissue properties are needed. We utilize a Transwell chamber with tunable collagen-hyaluronic acid (ColHA) hydrogels as an in vitro model to determine mass transport of macromolecules using non-invasive UV spectroscopy. Increasing hyaluronic acid (HA) concentration from 0 to 2 mg/mL within collagen gels decreases the mass transport of five macromolecules independent of size and charge and results in a maximum decrease in recovery of 23.3% in the case of bovine immunoglobulin G (IgG). However, in a pure 10 mg/mL HA solution, negatively-charged macromolecules bovine serum albumin (BSA), ß-lactoglobulin (BLg), dextran (Dex), and IgG had drastically increased recovery by 20-40% compared to their performance in ColHA matrices. This result was different from the positively-charged macromolecule Lysozyme (Lys), which, despite its small size, showed reduced recovery by 3% in pure HA. These results demonstrate two distinct regimes of mass transport within our tissue model. In the presence of both collagen and HA, increasing HA concentrations decrease mass transport; however, in the absence of collagen, the high negative charge of HA sequesters and increases residence time of positively-charged macromolecules and decreases residence time of negatively-charged macromolecules. Through our approach, ColHA hydrogels serve as a platform for the systematic evaluation of therapeutic macromolecule transport as a function of molecular characteristics.

Multi-feature-Based Robust Cell Tracking.

Cell tracking algorithms have been used to extract cell counts and motility information from time-lapse images of migrating cells. However, these algorithms often fail when the collected images have cells with spatially and temporally varying features, such as morphology, position, and signal-to-noise ratio. Consequently, state-of-the-art algorithms are not robust or reliable because they require manual inputs to overcome the cell feature changes. To address these issues, we present a fully automated, adaptive, and robust feature-based cell tracking algorithm for the accurate detection and tracking of cells in time-lapse images. Our algorithm tackles measurement limitations twofold. First, we use Hessian filtering and adaptive thresholding to detect the cells in images, overcoming spatial feature variations among the existing cells without manually changing the input thresholds. Second, cell feature parameters are measured, including position, diameter, mean intensity, area, and orientation, and these parameters are simultaneously used to accurately track the cells between subsequent frames, even under poor temporal resolution. Our technique achieved a minimum of 92% detection and tracking accuracy, compared to 16% from Mosaic and Trackmate. Our improved method allows for extended tracking and characterization of heterogeneous cell behavior that are of particular interest for intravital imaging users.

Fibronectin-Expressing Mesenchymal Tumor Cells Promote Breast Cancer Metastasis.

Tumor metastasis is connected to epithelial-mesenchymal heterogeneity (EMH) and the extracellular matrix (ECM) within the tumor microenvironment. Mesenchymal-like fibronectin (FN) expressing tumor cells enhance metastasis within tumors that have EMH. However, the secondary tumors are primarily composed of the FN null population. Interestingly, during tumor cell dissemination, the invasive front has more mesenchymal-like characteristics, although the outgrowths of metastatic colonies consist of a more epithelial-like population of cells. We hypothesize that soluble FN provided by mesenchymal-like tumor cells plays a role in supporting the survival of the more epithelial-like tumor cells within the metastatic niche in a paracrine manner. Furthermore, due to a lower rate of proliferation, the mesenchymal-like tumor cells become a minority population within the metastatic niche. In this study, we utilized a multi-parametric cell-tracking algorithm and immunoblotting to evaluate the effect of EMH on the growth and invasion of an isogenic cell series within a 3D collagen network using a microfluidic platform. Using the MCF10A progression series, we demonstrated that co-culture with FN-expressing MCF10CA1h cells significantly enhanced the survival of the more epithelial MCF10CA1a cells, with a two-fold increase in the population after 5 days in co-culture, whereas the population of the MCF10CA1a cells began to decrease after 2.5 days when cultured alone (p < 0.001). However, co-culture did not significantly alter the rate of proliferation for the more mesenchymal MCF10CA1h cells. Epithelial tumor cells not only showed prolonged survival, but migrated significantly longer distances (350 µm compared with 150 µm, respectively, p < 0.01) and with greater velocity magnitude (4.5 µm/h compared with 2.1 µm/h, respectively, p < 0.001) under co-culture conditions and in response to exogenously administered FN. Genetic depletion of FN from the MCF10CA1h cells resulted in a loss of survival and migration capacity of the epithelial and mesenchymal populations. These data suggest that mesenchymal tumor cells may function to support the survival and outgrowth of more epithelial tumor cells within the metastatic niche and that inhibition of FN production may provide a valuable target for treating metastatic disease.

Effect of hinge gap width of a St. Jude medical bileaflet mechanical heart valve on blood damage potential--an in vitro micro particle image velocimetry study.

The hinge regions of the bileaflet mechanical heart valve (BMHV) can cause blood element damage due to nonphysiological shear stress levels and regions of flow stasis. Recently, a micro particle image velocimetry (µPIV) system was developed to study whole flow fields within BMHV hinge regions with enhanced spatial resolution under steady leakage flow conditions. However, global velocity maps under pulsatile conditions are still necessary to fully understand the blood damage potential of these valves. The current study hypothesized that the hinge gap width will affect flow fields in the hinge region. Accordingly, the blood damage potential of three St. Jude Medical (SJM) BMHVs with different hinge gap widths was investigated under pulsatile flow conditions, using a µPIV system. The results demonstrated that the hinge gap width had a significant influence during the leakage flow phase in terms of washout and shear stress characteristics. During the leakage flow, the largest hinge gap generated the highest Reynolds shear stress (RSS) magnitudes (~1000 N/m²) among the three valves at the ventricular side of the hinge. At this location, all three valves indicated viscous shear stresses (VSS) greater than 30 N/m². The smallest hinge gap exhibited the lowest level of shear stress values, but had the poorest washout flow characteristics among the three valves, demonstrating propensity for flow stasis and associated activated platelet accumulation potential. The results from this study indicate that the hinge is a critical component of the BMHV design, which needs to be optimized to find the appropriate balance between reduction in fluid shear stresses and enhanced washout during leakage flow, to ensure minimal thrombotic complications.

Micro particle image velocimetry measurements of steady diastolic leakage flow in the hinge of a St. Jude Medical® regent™ mechanical heart valve.

A number of clinical, in vitro and computational studies have shown the potential for thromboembolic complications in bileaflet mechanical heart valves (BMHV), primarily due to the complex and unsteady flows in the valve hinges. These studies have focused on quantitative and qualitative parameters such as velocity magnitude, turbulent shear stresses, vortex formation, and platelet activation to identify potential for blood damage. However, experimental characterization of the whole flow fields within the valve hinges has not yet been conducted. This information can be utilized to investigate instantaneous damage to blood elements and also to validate numerical studies focusing on the hinge's complex fluid dynamics. The objective of this study was therefore to develop a high-resolution imaging system to characterize the flow fields and global velocity maps in a BMHV hinge. In this study, the steady leakage hinge flow fields representing the diastolic phase during the cardiac cycle in a 23 mm St. Jude Medical regent BMHV in the aortic position were characterized using a two-dimensional micro particle image velocimetry system. Diastolic flow was simulated by imposing a static pressure head on the aortic side. Under these conditions, a reverse flow jet from the aortic to the ventricular side was observed with velocities in the range of 1.47-3.24 m/s, whereas low flow regions were observed on the ventricular side of the hinge with viscous shear stress magnitude up to 60 N/m². High velocities and viscous shearing may be associated with platelet activation and hemolysis, while low flow zones can cause thrombosis due to increased residence time in the hinge. Overall, this study provides a high spatial resolution experimental technique to map the fluid velocity in the BMHV hinge, which can be extended to investigate micron-scale flow domains in various prosthetic devices under different hemodynamic conditions.