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

Mitral Valve Anatomy

Materials and Methods Dissection and freezing of porcine mitral valves

Mechanical Testing

Statistical Methods

•  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” Mechanical Testing Parameters

Results Frozen valves trend stiffer

Results Continued Fresh valves have more extensibility Significant differences in hysteresis

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 valve’s mechanical properties except in

hysteresis •  Freezing time between 1-12 weeks also has little effect on the valve’s mechanical

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

fresh tissue •  Heart valve research should be conducted on fresh samples

Future Objectives

•  Increase number of samples to get stronger statistical power •  Use this data to develop a cryopreservation technique that does not change the valve’s

mechanical properties

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.

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•  The mitral valve is located between left atrium and left ventricle and must close to resist systolic pressure

•  The mitral valve is constituted of two leaflets: anterior and posterior

•  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

•  The anterior rough zone, anterior clear zone and posterior leaflet differ in mechanical properties

•  The valve leaflets are anisotropic; stiffer in the circumferential direction than in the radial direction

A)Atrial view of the mitral valve. B) Dissected mitral valve split at a commissure. (Grande-Allen, 2004)

LV Chamber

“Vena Cava” “Aorta”

Compliance Chamber

Reservoir

Flow Meter

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

•  Dissect intact mitral valves from hearts

•  Fresh valves were stored in PBS at 4° C overnight

•  Frozen valves were soaked in either 50:50 Glycerol:PBS or 80:10:10 DMEM:BGS:DMSO

•  50:50 Gly:PBS at -20°C is the most common tissue freezing method •  DMSO is also commonly used as a cryo-protectant for cells

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

LV Chamber

“Vena Cava” “Aorta”

Compliance Chamber

Reservoir

Flow Meter

Clear

Posterior Rough

Sections used for mechanical testing

0 0.1 0.2 0.3 0.4 0.5 0.6 0.7−0.1

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Strain (mm/mm)

Stre

ss (M

Pa)

DataYoungs lineLinear RegionExtensibilityUltimate Stress/StrainToe RegionRadius of Curve

MVP

(Left) Data Analysis Summary. Young’s 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

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

•  Valves were measured for thickness and width under a stereomicroscope

^ p < 0.15

* p < 0.01

*

* p < 0.01

•  Valves were tested in uniaxial tension to evaluate Young’s modulus, extensibility, hysteresis, yield stress and strain, ultimate stress and strain, radius of curvature, and stress relaxation.

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

(Left) Extensibility is caused by crimp in collagen fibers. scale = 50µm Image from Liao, Acta Biomaterialia, 2005

^ ^ ^

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Section of valve tissue prepared for mechanical testing measured under stereomicroscope with image analysis

All error bars shown are standard error of the mean.

Less hysteresis represents more elastic behavior and less viscous behavior