Micro-patterned Hemodynamic Niches for Endothelial Regulation
of Coagulation on Prosthetic Card
Nadeem Khan, Shrinidhi Kanduru, Chris Frendl, Scott Tucker, Jonathan Butcher
Department of Biomedical Engineering, Cornell University, Ithaca, NY
BACKGROUND
OBJECTIVE
STATIC PLATELET ADHESION TEST
Valvular heart disease is prevalent in the U.S. with an
average of 5 million individuals diagnosed with it every
year.1 Two problems that occur with the aortic heart valve
are aortic stenosis and Regurgitation.2,3 Aortic stenosis is
the most prevalent valve disease in the aging population
and the third most common cardiovascular disease.4,5
Regurgitation occurs as a result of a weakened aortic
valve and allows backflow into the left ventricle. It
affects about 8% of women and 13% of men.6 How do
they propose to fix these problems? Valve replacements!
To functionalize the surface of the mechanical valve prosthesis to enable
endothelial-specific adhesion, effectively reducing the life-long usage of blood
thinners.
- Design chip to reduce shear and promote endothelial adhesion
- Assess cell viability on chip under shear
- Test platelet adhesion on chips with new design.
- Determine the presence of antithrombotic factors released from endothelial
cells
To test cell viability under high shear rates and platelet adhesion, PAVECs
were plated onto the chip with either fibronectin or collagen. Endothelial
cells were sheared in a bioreactor for 48 hrs at 200+dyne/cm2. A 5 minute
static platelet adhesion test was performed afterwards. The results are
shown in Figure 12 D-J.. Antithrombotic factors VE-vadherin, and eNOS
were also stained for.in Figure 12 A,B. The nucleus was stained using
Hoechst (Figure 12 C). The results of the experiment show that platelet
adhesion is reduced on chips coated with ligand and sheared endothelial
cells (Figure 12 I,J).
METHODS AND RESULTS
Figure 1: Aortic stenosis1
CLINICAL NEED
As of 2006, there were 104,000 valve related surgeries.7
Of those, there were 95,000 open heart surgeries for
valve replacement related stenosis.8 Two-thirds of current
valve replacements are mechanical in nature and tend to
last for 20-30 years.9,10 The other one-third is biological.
However they do not last as long as mechanical ones
with an average life span of 10-15 years.10
www.heart-valve-surgery.com
Figure 3: Mechanical heart
valve
PROBLEM
In a normal aortic valve, endothelial cells coat healthy cardiac systems and
prevent thrombus formation. Mechanical heart valves are missing these
endothelial lining. As a result:
-Shear forces cause platelet activation and red blood cells to rupture.
-There is an increase risk of thrombosis and clot formation.
-Patients need to take anticlotting medication for the rest of their lives.
Patients taking blood thinners have a difficult time clotting when injured.
Individuals with cardiac implants have a reduced quality of life due to their
requirement for a limited lifestyle. This includes inability to get pregnant, vast
reduction in physical activity, and high risk of bleeding. Coating the surfaces of
these implants with endothelial cells would prevent the need for anticoagulants
by creating a natural hemodynamic environment.
Blood flow
Endothelial Cells
Anti. Thromb. Factors
Blood Flow
(size relative to shear)
Red Blood Cell
Current problems in cardiology (2002)
GFOGER
SHEAR STRESS
GEOMETRY
ANALYSIS
Shear Rate (dynes/cm^2)
We use a peristaltic pump in conjunction with a
recently developed polycarbonate bioreactor to
simulate the pulsatile environment across the
cell-seeded surface of the MPVs. The reactor is
designed
to
enable
imaging
during
experimentation without requiring flow
disruption. Through fluorescent and confocal
microscopy we can test for the presence and
activity of cells after exposure to shear. For flatsurface mechanical valves, low shear rates
show highest cell adhesion with collagen
ligand.7
40
FN
FN-7
30
A
D
Platelets
PAVEC
E
F
Laminin
20
Control
VE-cadherin
10
.
B
0
50% Adhesion
Figure 7: Data showing the shear rate
found to detach 50% of adhered cells on
the flat surface after 10 min of flow using
six different ligands..
FOR
VARIOUS
WELL
Shear stress rates in the heart are about 40 times those tested to find the rate to
detach 50% adhered cells. To increase shear rates to match that of the heart and
keep cellular adhesion, wells were proposed (Figure 6). To identify the depth
and the width between each of the ‘corduroy’ walls, we examined two
dimensional flow analysis. Specifically, we identified the shear at the bottom of
our CAD developed wells based on depth and width alterations. Our testing
showed an overall correlation between all the data-points (Figure 8 and 9).
G
H
I
J
eNOS
C
Hoechst
Figure 12: (A) Antithrombotic
stains for VE-cadherin, eNOS (B),
and Hoechst (C) for 48 hr sheared
PAVEC cells.. Static platelet
Adhesion Test D-J. Platelets
(green) and endothelial cells (red).
Platelets adhered to the control
chip with no ligand (D); with both
ligands-collagen
(E)
and
fibronectin (F), respectively; on
chips
with
collagen
and
fibronectin,
respectively,
and
unsheared cells (G,H); and on
chips
with
Collagen
and
fibronectin, respectively; and with
collagen and fibronectin and
sheared cells (I, J).
CONCLUSION
We have been able to analyze shear stress along the surface of mechanical
heart valve and thereby develop a micro-patterned well chip designed to
*
reduce shear along the bottom of the wells, promoting endothelial cell
adhesion. Analysis of this micro patterned design has shown that epithelial
cells survive in wells for 48 hrs at 200+dyne/cm2 and release antithrombotic factors to help prevent platelet adhesion (Figure 12 I,J).
Well Properties Along Viable EC Shear Stress Margin
FUTURE PLANS
600
Spacing (um)
Figure 2: Regurgitation2
Coll
50
400
Below 7Pa
200
Above 7Pa
0
100
200
300
400
500
Width (um)
Figure 9: Optimization of spacing for 3-D
Figure 8: Shear stress measured at the bottom
of two dimensional wells for different depths
and widths.
D
channels with perpendicular flow. Shear stress
quickly increases above 7 Pa with a very low
margin as spacing increases.
Micro Pattern Chip Generation
From the preliminary models done (Figure 8 and 9) on shear stress for
optimization of well depth, width, and spacing, parameters for each criterion
were chosen and modeled in ANSYS to show shear stress at the bottom of the
well (Figure 10). A prototype chip was then made by CNF (Figure 11).
Figure 4: Thromboembolism rates for mechanical aortic
valves. The percentage rate of Point estimates of the
thromboembolism.
Future directions for our group include:
- The generation of multiple samples for our results so that we can prove
statistical significance
- Assess endothelial cell adhesion under shear stress of 1800 dyne/cm2 to
mimic actual shear within the heart.
- Whole blood viability
- The use of human endothelial cells to identify a viable seeding protocol
for patient implantation.
ACKNOWLEDGEMENT
We would like to acknowledge Butcher Lab: Jen Richards, Emily Howell,
Gretchen Mahler, and Russell Gould for their help; Mandy Esch for all of
her time and efforts in CNF making the prototype; and King’s Lab: Thong
Cao for his help in isolating Platelets for us.
REFERENCES
1. Boon et al. (2002) Heart 2.Atlas of Echocardiography yale.edu, 3. American Heart
Association, 4. Carabello et al. (2009) Lancet, 5. www.medicinenet.com, 6.
www.freemd.com, 7. Heart Disease and Stroke Stats. (2010) Circulation, 8.
www.ahrq.gov (2010), 9. Garver et al (1995) Prosthetic Heart Valve Epidemiology, 10.
Goldsmith et al. (2002) BMJ 11. Current Problems in Cardiology (2000)
Goldsmith et al. (2002)
BMJ
Figure 5: A thrombotic valve10.
Figure 6: A diagram depicting the idea of wells
with a geometry optimized for decreasing shear
stress at the bottom of the valve surface.
Figure 10: Ansys model of flow velocity over the
surface of the optimized well and corresponding
shear stress at the bottom of the wells.
Figure 11: Mechanical heart valve design.
Several etched wells to allow endothelial
attachment.