The Effect of Cryopreservative on Mechanical Properties of

Porcine Mitral Valve Leaflets

Sarah Danga, Dan Puperib, Patrick Connellb, and K. Jane Grande-Allen, PhDb
aDeBakey High School for Health Professions, Houston, TX; bDepartment of Bioengineering, Rice University, Houston, TX

Introduction
Motivation of Research
Mitral valves fail mechanically due to disease states such as mitral valve prolapse.
Cryopreservation is used to save tissue for later analysis, but freezing tissues may
change the mechanical properties of the tissue
This research evaluates the effects of different freezing mechanisms on the mechanical
properties of mitral valve tissue

Results Continued

Valves were tested in uniaxial tension to evaluate

Youngs modulus, extensibility, hysteresis, yield stress
and strain, ultimate stress and strain, radius of
curvature, and stress relaxation.

Fresh valves have more extensibility

MVP

* p < 0.01

5
2

Valves were tested in PBS bath at 37C (not pictured)

Mitral Valve Anatomy

Statistical Methods

The mitral valve is located between left atrium

and left ventricle and must close to resist
systolic pressure

n = 4
Data was analyzed using ANOVA with post-hoc Tukey HSD testing
* p < 0.01 defined for significance; ^ p < 0.15 defined as a trend

The mitral valve is constituted of two leaflets:

anterior and posterior

The anterior rough zone, anterior clear zone and

posterior leaflet differ in mechanical properties

Significant differences in hysteresis

Mechanical Testing Parameters

0.6

A)Atrial view of the mitral valve.

B) Dissected mitral valve split at a
commissure. (Grande-Allen, 2004)

The valve leaflets are anisotropic; stiffer in the

circumferential direction than in the radial
direction

0.5

Data
Youngs line
Linear Region
Extensibility
Ultimate Stress/Strain
Toe Region
Radius of Curve

0.2

0.1
0

Materials and Methods

0.1

0.2

0.3
0.4
Strain (mm/mm)

0.5

0.6

0.7

(Left) Data Analysis Summary. Youngs modulus is calculated to be the slope of the linear region during
which the collagen has uncrimped and is being pulled to failure. The extensibility is taken to be the linear
extrapolation of Young's line to the x intercept and represents the point in testing when the stress is being
balanced by collagen integrity rather than uncrimping. The ultimate stress and strain are measured to be the
point at which the valve integrity fails and it rips. (Right) Hysteresis is the area between the loading and
unloading curves and represents the energy lost during the process of loading and unloading tissue

Flow
Meter
LV Chamber

Dissection and freezing of porcine mitral valves

Obtain fresh porcine hearts from abattoir (Fisher Ham and Meats, Spring, TX)

(Left) Extensibility is caused by crimp in

collagen fibers. scale = 50m
Image from Liao, Acta Biomaterialia, 2005

Dissect intact mitral valves from hearts

Fresh valves were stored in PBS at 4 C overnight

Less hysteresis represents more elastic behavior and less viscous behavior

Discussion and Future Directions

Conclusions
Freezing changes the mechanical properties of valves
Valves in DMSO cryopreservative trend toward stiffer than Gycerol:PBS
Freezing temperature has little effect on the valves mechanical properties except in
hysteresis

Frozen valves were soaked in either 50:50 Glycerol:PBSCompliance

or 80:10:10
Reservoir DMEM:BGS:DMSO
Chamber

50:50 Gly:PBS at -20C is the most common tissue freezing method

Freezing time between 1-12 weeks also has little effect on the valves mechanical
properties

Vena Cava
Aorta

DMSO is also commonly used as a cryo-protectant for cells

Flow-196C
Valves were frozen for either one or 12 weeks at -20C, -80C or
Meter

Studies that use frozen tissue may not retain the same extracellular matrix arrangement as
fresh tissue

Results

LV Chamber

Mechanical Testing
Valves were prepared for mechanical testing by cutting circumferential sections of the
anterior leaflet (clear and rough zones) and posterior leaflet

0.3

Vena Cava
Aorta

0.1

Compliance Reservoir
Chamber

* p < 0.01

0.4
Stress (MPa)

The two leaflets differ in makeup. The posterior

leaflet and the rough zone of the anterior leaflet
are attached to chordae tendineae, while the
clear zone of the anterior leaflet does not attach
to any chordae

Frozen valves trend stiffer

Valves were measured for thickness and width under a stereomicroscope

Heart valve research should be conducted on fresh samples

^ p < 0.15

Future Objectives
^

Increase number of samples to get stronger statistical power

Use this data to develop a cryopreservation technique that does not change the valves
mechanical properties

Clear
Rough

Posterior

Sections used for mechanical testing

Acknowledgments
The authors would like to thank Dr. Grande-Allen and members of the Grande-Allen Lab for the
support and resources needed to conduct this research. Thanks to Dr. Lau at DeBakey High School
for guidance and support. Funding provided by NIH R01HL107765 grant and the NSF GFRP.

Section of valve tissue prepared for

mechanical testing measured under
stereomicroscope with image analysis
All error bars shown are standard error of the mean.