(692e) Aortic Smooth Muscle Cell Benchmark Force Measurement

Aortic Smooth Muscle Cell
Benchmark Force Measurement

Brian Koons¹

Julie A. Phillippi²

Thomas G. Gleason²

Amrinder S. Nain^1,3

¹Department of Mechanical
Engineering, Virginia Tech, Blacksburg, VA, USA

²Department
of Cardiothoracic Surgery, University of Pittsburgh, Pittsburgh, PA, USA

³School of Biomedical
Engineering and Sciences, Virginia Tech, Blacksburg, VA, USA

Introduction

Rupture of aortic aneurysm is the thirteenth leading cause of death in
the United States and is a clinical emergency. The main reason for the high
death rate is that the condition is commonly asymptomatic, until the aorta
ruptures, leading rapidly to death. An aortic aneurysm is characterized by an
enlargement of the aorta's diameter. 
Although vessel diameter varies among individuals, it can be related to
body surface area, and aortic diameter larger than 30 mm is concerning and
potentially life threatening [1]. Aortic aneurysm can be either fusiform,
affecting the entire circumference of the vessel, or saccular, where only a
portion of the circumference is enlarged. 
The dilatation is due to mechanical weakening of the blood vessel wall,
initiated by biological pathways that are distinct for aneurysms of the
thoracic and abdominal aorta.  Current expert
clinical opinion dictates surgical intervention to replace the aneurysmal aorta
with a synthetic graft when the maximal orthogonal aortic diameter reaches 50-55
mm. Unfortunately, the aneurysmal aorta has been known to dissect or rupture at
lower aortic diameters and accurate risk assessments for patients is
challenging and lacking. Hence, there is a great clinical need for enabling
technologies to improve rupture risk profiles, increased knowledge of the
biological mechanisms mediating the pathophysiology and new medical therapies
for patients affected by aneurysm.  We
have engineered a model system used to measure forces exerted by migratory primary
human aortic smooth muscle cells (HASMC). This technology could be used as a
benchmark to determine putative differences in cell forces exerted by diseased HASMCs.
 Using our previously reported STEP
(Spinneret based Tunable Engineered Parameters) non-electrospinning fiber
fabrication platform [2], aligned polymeric fibers were deposited in multiple perpendicular
layers and forces exerted by migratory cells were calculated through the
deflection of fibers in the elastic limit using Euler beam mechanics. This
platform will provide fresh insights in single-cell force generation in normal
vs. diseased cells and can potentially be used to determine efficacy of novel
therapeutics to treat patients at risk for aortic catastrophe.

Materials and Methods

STEP manufactured nanofibers were suspended in a highly aligned and
oriented fashion on polystyrene frames. Various concentrations of polystyrene
dissolved in xylene was used to fabricate fibers of
250 nm and 2 𝜇m,
respectively. The two fiber types were oriented perpendicular to one another
and fused at intersections to engineer a nanofiber mesh where both fiber
diameters coexist within the same 2-D geometric plane. The fibers were coated
with the extracellular matrix protein fibronectin (2 𝜇g/ml) to increase cell attachment (seeding
density 20,000 cells/ml). HASMCs were isolated
from non-aneurysmal patients with approval of the IRB and with informed patient
consent. Time-lapse phase contrast microscopy and forces measurements were
conducted using Carl Ziess microscope and Axiovision software. As cells migrated
on the fibers, fiber deflections were observed and measured to calculate the
cell forces using Euler beam theory.

Results

Cellular force generation was found to be dependent upon fiber
structural stiffness (N/m). The cells were unable to deflect the stiffer 2 𝜇m diameter fibers. However,
large deflections, ranging from 2 to 12 𝜇m, occurred on the less stiff 250
nm fibers. Therefore, the less stiff fibers can be modeled as deformable beams
where the stiff fibers at their edge create a simply supported boundary
condition. Preliminary results with HASMC exhibited a force generation of 0.01
nN at the least stiff regions along the fiber and up to 0.8 nN
near the stiffest regions of the fiber, as shown in Figure 1. These
measurements provide the benchmark values for non-diseased HASMC lines and can
be used to compare force generation by cells from diseased patients.

Figure 1. (a) Healthy HASMC
deflecting 250 nm fiber segments between stiff boundaries, (b) immunocytochemistry
for the cytoskeletal proteins F-actin (red) and zyxin (green) while a healthy
HASMC is deflecting a fiber,  and (c) healthy
HASMC force generation on fibers of various stiffness.

Discussion

The STEP fiber fabrication technique provides a novel biophysical platform
for determining the force generation of single migratory HASMCs. The method may
be used to establish a new force-based hypothesis for aneurysm development,
progression and risk for rupture and will help test the efficacy of novel
therapeutics.  It is expected that future
studies on diseased cells will show a decrease in cell force generation for  HASMC isolated
from aneurysmal patients when compared with healthy HASMC. This enabling
technology can later be applied for the study of several other pathologies.

References

[1] Sakalihasan, N Limet, R Defawe, O D. Abdominal aortic
aneurysm. Lancet. 2005

 [2] Nain AS,
Sitti M, Jacobson A, Kowalewski T, Amon C. Dry Spinning Based Spinneret Based
Tunable Engineered Parameters (STEP) Technique for Controlled and Aligned
Deposition of Polymeric Nanofibers. Macromolecular Rapid Communications.
2009;30:1406-12.