Abstract Hemodynamic forces play an important role in

maintaining the function and structure of blood vessels. In this

work we discuss the design of an in vitro flow loop mimicking

the aortic arch that is aimed at studying the response of

endothelial cells to geometric curvature and the resulting flow

field. The flow system allows for quantitative flow visualization

measurements, and for access to the vessel lumen for cell

culturing and sampling.

I. INTRODUCTION

Cardiovascular disease such as atherosclerosis is a common disease in developing and developed nations affecting the circulatory system. Its economic and social impacts are enormous [1] and significant efforts have been directed at improving its outcomes. This type of disease is affected by a large number of interconnected factors such as nutritional patterns, exercise and physical activity, and hereditary qualities.

Recently, studies have suggested that the interconnectedness between hemodynamic factors (e.g. flow shear and pressure) and physiological processes (e.g. inflammation) plays an important role in the development and progress of atherosclerosis and other cardiovascular diseases [2]. This interconnectedness or interplay is referred to as mechano-transduction in which the flow forcing induces alterations at the molecular level of blood vessel components such as endothelial and smooth muscle cells. These changes, whether temporary or chronic, induce the release of certain bio-chemical and inflammatory agents that in turn modify the function and structure of the endothelial lining [3]. The formation of plaque is one manifestation of such chronic modifications. In vitro investigations have been essential in the understanding of cardiovascular disease through identifying certain mechano-transduction pathways in health and disease states.

*Research supported by the Faculty of Engineering and Architecture

M.A. Alloush, undergraduate student, Mechanical Engineering Dept., Faculty of Engineering and Architecture, AUB, Beirut, Lebanon (e-mail:

mha56@aub.edu.lb). G. F. Oweis, associate professor, Mechanical Engineering Dept., Faculty

of Engineering and Architecture, AUB, Lebanon (phone: +961-1-350000

Ext. 3596/3623; fax: +961-1-44477; e-mail: goweis@aub.edu.lb). R. Nasr, assistant professor, Dept. of Anatomy, Cell Biology, and

Physiological Sciences, Faculty of Medicine, AUB, Beirut, Lebanon (e-

mail: rn03@aub.edu.lb). A. Zeidan, assistant professor, Dept. of Anatomy, Cell Biology, and

Physiological Sciences, Faculty of Medicine, AUB, Beirut, Lebanon (e-

mail: az29@aub.edu.lb).

In vitro studies [4, 5] are advantageous as they provide the ability to individually isolate and study cardiovascular factors by minimizing interferences from many other complicating factors present in vivo. In vitro inflammation studies of the cardiovascular system have probed a large number of possible disease-affecting factors such as hypertensive pressure, pulsatile pressure, mechanical stretch, temperature, oxygen level, and nutrient supply [6,7,8].

In this work we are particularly interested in the aortic arch geometry for studying inflammatory agents affecting the endothelial cells lining the vessel lumen. The cells grown on the aortic arch lumen will be subjected to differing hemodynamic forcing and shear stress (disturbed/ undisturbed flow patterns) depending on which part of the aorta they happen to grow on (closer to the inlet or outlet / inner or outer wall of the bend); see Figure 1. The curved

characterized by flow detachment and low shear stress on the

as it proceeds to negotiate the curved flow path. The flow on the outer bend wall remains attached with a higher, healthy level of shear stress. This will give rise to varying bio/chemical secretions or signaling (e.g. nitric oxide) and growth patterns of the cells. A primary aim of this work is to produce qualitative and quantitative mechano-transduction correlations between the hemodynamic forcing and the biochemical cell signaling through obtaining detailed shear stress distribution estimates in the aorta, and by quantification of the cellular response in various parts of the aorta.

We discuss an aortic flow loop design to fulfill two requirements: i) optical access to the arterial vessel interior to allow quantitative flow visualization (particle image velocimetry, PIV) and hemodynamic flow properties estimation; and ii) physical access to the aortic lumen to permit cell culturing and growth under flow conditions. The aorta model is produced by first manufacturing two halves of a mold preserving the negative impression of the aortic arch. The mold is then filled in two steps with liquid PDMS (poly-dimethyl-siloxane) to solidify. PDMS is expected to have good cell adhesion properties, and it is optically clear. The result is a two-piece, transparent aortic bend vessel, with the ability to disconnect the two pieces for access to the vessel lumen. The aortic model is coupled to a circulating flow system to drive and control the aortic flow parameters.

II. CIRCULATING FLOW LOOP

A drawing of the aortic flow system is shown in Figure 1. Flow circulation into the aortic arch is maintained by a peristaltic pump

An Aortic Arch Flow Loop for the Study of Hemodynamic-Induced

Endothelial Cell Injury and Inflammation*

Mhamad M. Alloush, Ghanem F. Oweis, Rihab Nasr, and Asad Zeidan

2014 Middle East Conference on Biomedical Engineering (MECBME)February 17-20, 2014, Hilton Hotel, Doha, Qatar

978-1-4799-4799-7/14/$31.00 ©2014 IEEE 67

to pulse dampers. The temperature of the working fluid is monitored and maintained at for optimal endothelial cell viability. The flow leaving the pump enters a flow straightener section, followed by a development-length straight pipe piece to generate a developed flow entering the aortic bend. The development length pipe has a flush connection with the aorta. A static pressure transducer is used to monitor the system pressure. The circulatory system components and the salient design considerations are discussed below.

Figure 1. Above: Schematic of the aortic arch circulating flow loop system

showing the aortic arch, pump, inlet/oulet pulse dampers, and the magnetic flow meter. Below: the bottom part of the aortic arch PDMS cast with the

A. The Aortic Bend

The aorta model has a 14.3 mm ID round cross section, and the bend lies in a plane with the inlet and outlet separated by . The radius of curvature at the inner wall is 35 mm. These dimensions are derived from typical physiological values [9, 10]. The model is manufactured in two parts to

The PDMS used for the aorta cast has very good cell adhesion qualities and it is optically clear for flow visualization and imaging. However, the PDMS index of refraction is 1.43, which would induce significant image distortions if care is not taken with the selection of the working fluid. The fluid should have a matched index of refraction with the PDMS; and it should

have a biologically relevant kinematic viscosity to

result in a matched range of hemodynamic forcing, which

requires matching the flow Reynolds number; or alternatively the Dean number that derives from it and used specifically for curved vessels. There are a number of alternatives that can be used to satisfy both requirements including aqueous solutions of sucrose, sodium chloride, sodium iodide, glycerol, or combinations thereof at various concentrations. These provide sufficient, simultaneous control over the index of refraction and the kinematic viscosity. The mechanical and biological measurements will be conducted separately and they are not to be performed simultaneously because the cells will block the optical path needed for flow visualization; while some of the aforementioned working fluids can be toxic to the cells and/or will produce un-intended biochemical stimulants.

For the biological arm of the study, it is necessary to be able to sterilize the aortic model for cell culturing. Steam autoclaving has been successfully attempted without altering neither the optical quality nor the dimensional tolerances. Moreover, the real-life size of the vessel (that of a major artery) will enhance the prospects of detecting biochemical signaling factors from various groups of cells growing on different parts of the aortic arch that are associated with different hemodynamic patterns. This gives rise to a range of shear stress levels depending on the relative spatial location (inlet region, mid-way inner bend wall, mid-way outer bend wall, outlet region)

B. Circulating Pump, Pulse Dampers, and Flow Rate

The choice of peristaltic pump was constrained by the need to maintain sterility of the wetted parts of the flow loop. However, peristaltic pumps typically give rise to pressure pulsations particularly at lower rotational speeds. Pulsatility is known to influence cell signaling [11]. Two pulse dampers will be installed at the pump inlet and outlet. They work to insulate the pumloop. The intention in having a steady flow is to narrow down the range of variables in the experiments to probe the effect of the curved geometry flow on the endothelial cells. Adding Pulsatility or other forms of pressure variations, although important, can interfere with the geometric effect and would add more complexity to the system and to the interpretation of results. Moreover, other studies have shown that the effect of shear stress can be studied under static pressure and steady flow conditions [12, 13].The pulse dampers are constructed with partially filled inverted flasks and insertion tubes. The simple, but effective construction exploits the large acoustic impedance mismatch between water and the air gap in the upper part of the flask. A static pressure transducer will monitor any pressure fluctuations about the mean, but critically the mean static pressure. The compliance present in the system and tubing makes this step necessary. A venting valve combined with pressurizing syringe action will be used to set the mean static arterial pressure. In addition to their pulse damping function, the flasks will be helpful in providing a sterile, mess-free filling and emptying process. By setting the flasks upright at the end of the experiment, the circulating fluid will collect in them and it can be taken for processing and biochemical analyses.

Although the pump has a flow rate indicator it is advisable to calibrate it or have an independent flow

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measurement. This is provided by a magnetic flow meter installed in the flow loop.

C. Heaters

Electric heating bands will be wrapped around the pulse damper flasks to maintain a fluid temperature of monitored by a thermocouple. The temperature will be maintained by increasing the percentage of power input to the heaters in steps until it equalizes the heat loss from the flow loop at . The temperature will stay steady once this sweet spot is reached. This is thought to be a simpler and superior solution (as long as the lab temperature remains constant) over a proportional-integral-derivative feedback controller where tuning is necessary and the temperature can continually fluctuate by as much as and induce undesired thermal stressing on the cells.

D. Inlet Development Length Piece

Downstream from the flow meter, the fluid enters the development length piece. It is a straight pipe of length L and diameter D the same diameter as the aortic arch (ID 14.3 mm) and is flush mounted with it. It contains an upstream flow straightener section formed of >80 small stainless pipes 1.27 mm ID, and 20 mm long. They act to streamline the flow, set it unidirectional, and break up any large-scale vortical structures. The length ratio L/D > 10 provides a stretch of pipe for the flow to sufficiently develop prior to entering the aorta. Minimizing the total working fluid volume was an important consideration aiming at maximizing the biochemical signaling concentration detection sensitivity.

III. PIV FLOW VISUALIZATION

PIV is the primary flow measurement tool used in this study to characterize the hemodynamic forcing in the aortic arch. PIV works by seeding tracer particles into the flow and illuminating a 2-D planar area in the flow with a laser sheet. The seeded particles will appear as bright points in an otherwise dark background. A camera is used to take time-lapsed images of the flow tracers as they move with the flow. A cross correlation algorithm analyzes consecutive image pairs and produces 2-D maps of the tracer particle displacements and velocities at that instant of time. Further analysis of the velocity fields can provide quantitative hemodynamic flow properties, with the most important being the shear stress; which is the friction force exerted by the blood flow on the endothelial cells. Additionally, PIV can provide information on the level of flow disturbance and turbulence in different parts of the aortic arch, and it can provide evidence on the locations of flow separation and low shear stress, often associated with vessel disease. These are all critical factors affecting endothelial cell health and function.

Figure 2 shows a schematic of the PIV implementation with two different laser light sheet orientations. The camera (not shown) will be oriented perpendicular to the light sheet to image the seed particles illuminated in the laser sheet. It is possible to orient the laser light sheet to measure the 2-D velocity vector distribution in a cross sectional plane of the aortic arch . This would permit the visualization of the out of plane vorticity in the axial direction taken along the center of the aortic arch. It is possible to orient the laser sheet to measure in the plane; in the same plane

containing the bend to estimate the shear stress at the inner wall and at the outer wall. This particular measurement setting will be critical in identifying regions of separated flow and low shear stress, as well as regions with normal shear stress. Low shear stress regions are generally associated with disease. It is possible to do other planes at different angular orientations as needed.

An important parameter in the PIV experiments is the spatial resolution of the acquired data. To be able to accurately measure wall shear stresses and wall velocity gradients the PIV system should have sufficient resolution. This parameter can be readily changed in our acquisition system through setting the image magnification factor. Our system s spatial resolution can be as low as 0.1 mm. Moreover, near wall laser reflections in the PIV images can act to reduce the signal-to-noise ratio and can cause increased uncertainties in the wall velocity gradient. Index of refraction matching of the working fluid should take care of significant part of this issue. The use of fluorescent particles with selective reflected light filtering can be an additional measure in improving the near wall measurements.

Figure 2. Two different PIV measurement planes produced by changing the laser sheet orientation into two orthogonal directions (laser sheet A) and

(laser sheet B)

IV. ENDOTHELIAL CELL ADHESION AND GROWTH

Endothelial cells cover the interior walls of the circulatory

system and they come in direct contact with the blood flow.

Endothelial cells will align themselves in the direction of the

blood flow. While in regions where they are chronically

from flow detachment, these cells will grow in unorganized

patterns.

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Figure 3. Below: One of the available setups for the microscopic imaging

of the endothelial cells in the PDMS aortic arch model. Above: A sample

image at 40X confirming that the (low seeding) cells have attached well to the aortic arch model, 24 hours after the seeding process.

It is critical for the progress of this investigation that

endothelial cell adhere well and grow on the lumen of the

PDMS aortic model. Indeed, Figure 3 is a microscope

picture of the endothelial cell culture, and, although the

initial seeding density was quite low, it provides visual

confirmation of the viability of the cells on the lumen, and

their ability to adhere, multiply, and branch out.

Optimization experiments to determine the optimal

concentration for seeding and culturing of the cells in this

setup are currently underway. This is contrasted

to typical hemodynamic-induced cell injury studies which

focus on simpler geometries such as petri dishes under

various flow and motion patterns.

V. CONCLUSION AND FUTURE WORK

A circulatory system to study flow-induced endothelial

cell injury and inflammation in the aortic arch geometry was

discussed. This investigation is unique for the study of

cardiovascular disease because it bridges an important gap

between in vitro studies in unrealistically simple geometries

(mostly petri dishes), and in vitro studies performed on

realistically complex geometries (excised blood vessels from

rats). It was shown that the proposed system has met the

primary requirements of i) transparency for flow

visualization and imagery, and ii) biocompatibility for cell

growth and viability. Our next step is to perform detailed

PIV characterization of the hemodynamic flow properties in

the aorta and map the regions of disturbed and undisturbed

flow for a range of flow rates indicative of rest and exercise.

These findings will be used to predict regions of cell injury.

Direct correlations between the flow patterns and cellular

response will be sought.

ACKNOWLEDGMENT

We thank Joseph Nassif, George Jurdi, and Joseph

Zullikian from the FEA machine shop for their help in

constructing the aorta mold.

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