Physical Forces and the Mammalian Cell

400 Pages·1993·6.938 MB·English

by  John A. Frangos (Eds.)

PHYSICAL FORCES
AND THE
MAMMALIAN CELL
Edited by
John A. Frangos
Department of Chemical Engineering
Pennsylvania State University
University Park, Pennsylvania
ACADEMIC PRESS
HARCOURT BRACE JOVANOVICH, PUBLISHERS
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This book is printed on acid-free paper. ©
Copyright © 1993 by ACADEMIC PRESS, INC.
All Rights Reserved.
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Library of Congress Cataloging-in-Publication Data
Physical forces and the mammalian cell / edited by John A. Frangos
p. cm.
Includes bibliographical references.
ISBN 0-12-265330-0
1. Biophysics. 2. Cell physiology. I. Frangos, John A.
QH505.P46 1993
599'.087-<lc20 92-23514
CIP
PRINTED IN THE UNITED STATES OF AMERICA
92 93 94 95 96 97 EB 9 8 7 6 5 4 3 2 1
CONTRIBUTORS
Numbers in parentheses indicate the pages on which the authors' contributions begin.
Albert J. Banes (81), Department of Surgery, University of North Carolina at
Chapel Hill, Chapel Hill, North Carolina 27599
Frangois Berthiaume (139), Department of Chemical Engineering, Pennsylvania
State University, University Park, Pennsylvania 16802
Rudi Busse (223), Department of Applied Physiology, University of Freiburg,
D-7800 Freiburg, Germany
Peter F. Davies (125), Department of Pathology, Pritzker School of Medicine,
University of Chicago, Chicago, Illinois 60637
Randal O. Dull (125), Department of Pathology, Pritzker School of Medicine,
University of Chicago, Chicago, Illinois 60637
John A. Frangos (139), Department of Chemical Engineering, Pennsylvania State
University, University Park, Pennsylvania 16802
1
Peggy R. Girard (193), Biomechanics Laboratory and School of Mechanical
Engineering, Georgia Institute of Technology, Atlanta, Georgia 30332
Linda Hansen (61), Department of Surgery, Children's Hospital, Harvard Medical
School and Department of Pathology, Brigham and Women's Hospital, Boston,
Massachusetts 02115
Gabriel Helmlinger (193), Biomechanics Laboratory and School of Mechanical
Engineering, Georgia Institute of Technology, Atlanta, Georgia 30332
Donald Ingber (61), Department of Surgery, Children's Hospital, Harvard Medical
School and Department of Pathology, Brigham and Women's Hospital, Boston,
Massachusetts 02115
Seth Karp (61), Department of Surgical Research, Children's Hospital, Boston,
Massachusetts 02115
B. Lowell Langille (249), Vascular Research Laboratory, Toronto Hospital and
Department of Pathology, University of Toronto, Toronto, Ontario M5G 2C4,
Canada
1
Present address: School of Biology, Georgia Institute of Technology, Atlanta, Georgia 30332.
xiii
xiv Contributors
Larry V. Mclntire (275), Cox Laboratory for Biomedical Engineering, Institute of
Biosciences and Bioengineering, Rice University, Houston, Texas 77251
James D. Michaels (291), Department of Chemical Engineering, Northwestern
University, Evanston, Illinois 60208
David Mooney (61), Department of Surgical Research, Children's Hospital and
Department of Chemical Engineering, Massachusetts Institute of Technology,
Cambridge, Massachusetts 02115
Robert M. Nerem (193), Biomechanics Laboratory and School of Mechanical
Engineering, Georgia Institute of Technology, Atlanta, Georgia 30332
Eleftherios T. Papoutsakis (291), Department of Chemical Engineering, North­
western University, Evanston, Illinois 60208
George Plopper (61), Program in Cell and Developmental Biology, Harvard Medi­
cal School, Boston, Massachusetts 02115
Ulrich Pohl (223), Department of Physiology, University of Lubeck, Lubeck,
Germany
Sridhar Rajagopalan (275), Cox Laboratory of Biomedical Engineering, Institute of
Biosciences and Bioengineering, Rice University, Houston, Texas 77251
2
Paul Todd (347), Chemical Science and Technology Laboratory, National Insti­
tute of Standards and Technology, Boulder, Colorado 80303
Roger Tran-Son-Tay (1), Department of Mechanical Engineering and Materials
Science, Duke University, Durham, North Carolina 27706
2
Present address: Department of Chemical Engineering, University of Colorado at Boulder,
Boulder, Colorado 80309.
PREFACE
It is increasingly evident that mechanical forces play a crucial role in
the physiology of living systems. Over the past fifteen years there has been
exponential growth in the research in understanding how mechanical
forces act on various tissues. It is now known that mechanical forces
regulate vascular diameter and caliber, bone remodeling, hearing, gastroin­
testinal motility, and skin growth. It appears that many of the tissue
responses and mechanisms have some degree of commonality; however, a
comprehensive and unifying review of these aspects is lacking.
The objective of this text is not only to present reviews of the current
state of knowledge, but also to provide guidelines and techniques for
further research. We focus here on how mechanical forces, such as
hydrodynamic shear and elongational strain, play a role in the normal
physiology of the cardiovascular system, blood cells, bone, and other
mammalian tissues. The mechanisms of how such forces act on cells are
discussed. In addition, the implications of physical forces to bioreactor
design and tissue engineering are examined. The intended audience for this
text is cardiovascular physiologists, hematologists, and bioengineers.
The chapters have been arranged in the order of methodology, theoret­
ical mechanisms, basic studies on cultured cells, vascular physiology, and
biotechnology applications. Chapter 1 discusses and evaluates the various
experimental techniques and devices that can be used to study the role of
mechanical forces on cells and tissues. The second chapter deals with a
proposed mechanism of how hydrodynamic shear affects cells, with partic­
ular emphasis on endothelial cells. Chapter 3 reviews what is known about
how elongational strain affects cells of various tissues. Chapters 4, 5, and 6
address how hydrodynamic shear affects cells, with particular emphasis on
endothelial cells. Chapters 7 and 8 address the acute and chronic effects of
blood flow on vascular physiology. Flow effects on blood cells and other
suspended cells are discussed in Chapter 9. Chapter 10 deals with the
importance of hydrodynamic forces in the design and operation of bioreac­
tors. Chapter 11 deals with the role of gravity in the context of hydrody­
namic forces.
The importance of understanding how mechanical forces regulate
mammalian cell and tissue function is demonstrated by the exponential
growth of research in the field. This timely text, contributed by a diverse
group of pathologists, biochemists, and bioengineers, comprehensively
examines mechanotransduction and serves as a convenient resource for
the design of new research.
xv
CHAPTER 1
Techniques for Studying
the Effects of Physical Forces on
Mammalian Cells and Measuring
Cell Mechanical Properties*
Roger Tran-Son-Tay
I. INTRODUCTION
Enormous progress has been made in the last decade toward under­
standing the effects of physical forces on the structure and function of
mammalian cells. However, the mechanisms by which shear stress and
strain modulate cell morphology and metabolism are still not well under­
stood. An understanding of the effects of shear stress and strain can be
achieved through studies in which cell populations are exposed to con­
trolled and well-defined flow or deformation environments. Very often, the
results reported in the literature are contradictory because of the different
methodologies used. For example, the type of cell, the substrate, the
medium, and the nature of the flow environment or deformation must all
be "controlled" in order to rationalize the behavior of a given cell.
The kind of experimental technique used depends largely on the type
of cells and properties to be studied, and, in general, techniques are
classified accordingly. However, consistent with an approach that focuses
*This work was supported by the National Institutes of Health through grant 2R01-HL-23728
and the National Science Foundation through grant BCS-9106452.
PHYSICAL FORCES Copyright © 1993 by Academic Press, Inc.
1
AND THE MAMMALIAN CELL All rights of reproduction in any form reserved.
2
Roger Tran-Son-Tay
on the mechanical effects, experimental techniques will be classified here in
terms of the nature of the physical force used to deform the cells. For
example, techniques that load the cell by the action of hydrodynamic force
are distinct from techniques that specifically cause membrane stretch.
The objectives of this chapter are to review the techniques used to
study the effects of fluid flow and strain on mammalian cells and to discuss
methods that allow mechanical properties to be determined. A range of
instruments provide well-defined flow fields, and cells can be strained by
flow or surface stretch deformations. Some of the most common instru­
ments used for studying suspended and/or attached cells are described,
and some of the problems that arise when using these instruments are
outlined. It is my intention to address some questions that have been
inadequately treated, and to illustrate the specific kinds of problems that
are associated with each technique so that readers can make knowledgeable
choices for their own particular applications.
II. FLOW DEFORMATION
The focus of this section is on instruments that produce well-defined
flows. Turbulent flow effects and their relevance to cell cultures will be
discussed in the next section. Many of the fluid shear devices were
originally developed to study the rheological properties of suspensions and
associated deformations of the individual suspended particles. In order to
discuss the deformation produced by flow for a given cell, we must
recognize that two general classes of culture cells are usually identified: (1)
anchorage-dependent cells, which require attachment to a solid substrate
for normal growth; and (2) suspension cells, which are able to grow freely
suspended in the culture medium. The possible influence of hydrodynamic
forces on a given cell is clearly dependent on whether it is attached to a
substrate or freely suspended.
The present section introduces the fundamental concepts and assump­
tions needed for analysis of fluid motion. In the forthcoming sections the
suspending fluid is assumed to be
1. A continuum. The fluid is treated as an infinitely distortable and
divisible substance. The continuum assumption is valid in treating the
behavior of fluids under most conditions. A fluid particle is just a linguistic
convenience for specifying an arbitrarily small fluid element.
2. Incompressible. Flows in which variations in density are negligible
are termed incompressible (i.e., the density remains constant throughout
3
1 • Techniques for Studying Physical Forces
the volume of the fluid and throughout its motion). In other words, there
is no noticeable compression or expansion of the fluid. It may be easy to
compress fluids with static devices, but it is very difficult to get compres­
sion through flow. The fluid behind pushes on the fluid in front, and the
latter flows rather than compresses. In fluid dynamics, the question of
when the density may be treated as constant involves more than just the
nature of the fluid (i.e., liquid or gas). Actually, it depends mainly on a
certain flow parameter (the Mach number). We then speak of incompress­
ible and compressible flows, rather than incompressible and compressible
fluids. Most liquid flows are essentially incompressible. However, water
hammer and cavitation are examples of compressible effects in liquid flows.
Whenever a valve is rapidly closed in a pipe, a positive-pressure wave is
created upstream of the valve and travels at the speed of sound. This
pressure may be great enough to cause pipe failure. This process is called
water hammer. Cavitation occurs when vapor is formed at those points in
the flow field where the local pressure falls below the saturation pressure;
there, local boiling takes place without the addition of heat. For most
gases, compressibility effects are negligible up to a Mach number Ma of
about 0.3 (the maximum density variation is less than 5% for values of
Ma < 0.3). The Mach number is defined as the ratio of the flow speed to
the local speed of sound. The speed of sound in air at sea level under
standard conditions is about 340 m/s, so that air behaves nearly as an
incompressible fluid for speeds of up to about 113 m/s. For comparison,
the speed of sound in water is about 1500 m/s.
3. Newtonian. The fluid exhibits a linear proportionality between the
applied shear stress, and the resulting rate of deformation obeys Newton's
law of viscosity. Water, the fluid of main concern here, is virtually a perfect
Newtonian fluid.
The basic equations that enable us to predict fluid behavior are the
equations of motion and continuity. The equations of motion for a real
fluid may be developed from consideration of the forces acting on a small
element of fluid, including the shear stress generated by fluid motion and
viscosity. The derivations of these equations, called the Navier-Stokes
equations, are beyond the scope of this chapter, and are listed for future
reference. The equations of continuity are developed from the general
principle of conservation of mass, which states that the mass within a
system remains constant. The term real fluid means that we are dealing
with situations in which irreversibilities (e.g., friction) are important.
Viscosity is the fluid property that causes shear stresses in a moving fluid; it
is also one way by which irreversibilities or losses are developed. Without
viscosity in a fluid, there is no fluid resistance.
4
Roger Tran-Son-Tay
For incompressible fluid with constant viscosity μ, the equations of
motion or Navier-Stokes equations are
density, μ is the fluid dynamic viscosity (commonly referred as viscosity), t
2
is time, D/Dt is the material or total time derivative, and V and V are the
nabla and Laplace operators, respectively (Happel and Brenner, 1986).
Simple flow experiments performed by Newton led him to two conclu­
sions concerning fluid friction that are fundamental to all that is known of
the mechanics of real fluids. The first comes from the observation that a
fluid does not slide along a solid boundary surface, but rather adheres to it
in all cases; This implies that the tangential flow component at a solid
boundary vanishes (no-slip condition). In addition, of course, one must
satisfy the kinematical condition that the normal velocity of the fluid is the
same as that of the boundary. This latter condition holds regardless of
whether the surface is fluid or solid, or whether the fluid is viscous. The
second conclusion deduced by Newton relates to the force exerted by a
fluid and a solid boundary surface on each other. He found, with his
one-dimensional flow experiment, that the shear stress τ is linearly propor­
tional to the shear rate y. For any flow field V = V(x, y, z, t), and
incompressible Newtonian fluid is defined as
τ = μy (2)
where τ is the stress tensor and γ is the rate of deformation tensor. The
proportionality factor μ is the dynamic viscosity of the fluid and is
independent of the flow geometry.
Viscous flow regimes are classified as laminar or turbulent on the basis
of the internal flow structure. In the laminar regime, the fluid moves in
layers, or laminas, one layer gliding smoothly over an adjacent layer with
only a molecular interchange of momentum. Any tendencies toward insta­
bility and turbulence are damped out by viscous shear forces that resist
relative motion of adjacent fluid layers. Flow structure in the turbulent
regime is characterized by random three-dimensional motions of fluid
particles superimposed on the mean motion. The nature of the flow, that
is, whether laminar or turbulent, is determined by the value of a dimen-
sionless parameter, the Reynolds number Re
pVL VL
Re =
ν
5
1 • Techniques for Studying Physical Forces
where V is the fluid velocity, L is a characteristic length descriptive of the
flow field, and ν is the fluid kinematic viscosity. It is not the dynamic
viscosity and the density that matter so much in the determination of the
nature of the flow but their ratio: the higher the kinematic viscosity, the
lower the Reynolds number.
Instruments are usually designed to provide simplified and well-defined
flow fields to facilitate the analysis and interpretation of the data. For this
reason, flows generated by the fluid shear devices described in this section
are assumed to satisfy the following conditions (in addition to the contin­
uum, incompressible, and Newtonian conditions mentioned above):
(4) laminar,
(5) steady, and
(6) fully developed.
These conditions and those stated earlier will be referred to as assumptions
(1-6) in the text. Laminar flow is defined above. Steady flow occurs when
conditions (e.g., velocity, density, pressure, and temperature) at any point
in the fluid do not change with time. A flow is fully developed when the
shape of the velocity profile no longer changes with increasing distance
from the flow entrance. The flow may approach full development asymp­
totically, but it is said to be fully developed for practical reasons when it
has achieved 99% of the final axial velocity. The distance downstream
from the entrance to the location at which fully developed flow begins is
called the entrance length.
There is no universally accepted classification of flow dimension, and
consequently some clarification is necessary. A flow is commonly classified
as one-, two-, or three-dimensional depending on the number of space
coordinates required to specify the velocity field only. However, some
investigators choose to classify a flow as one-, two-, or three-dimensional
on the basis of the number of space coordinates required to specify all
fluid properties. For this reason, the exact same flow field is sometimes
defined as one-dimensional by one group and two-dimensional by another.
For example, the laminar flow in a parallel-plate chamber is defined as
one-dimensional by Truskey and Pirone (1990), whereas Levesque and
Nerem (1989) defined it as two-dimensional; the velocity field is a function
of the vertical distance y only, and the pressure distribution is a function
of the axial distance χ (Fig. 1). Whatever the classification, the motion of a
fluid is completely determined when the velocity vector is known as a
function of time and position. Knowledge of the velocity field permits
calculation of the pressure distribution, shear stresses, and flow rate. The
location of an individual particle is given by position coordinates through
integration of the velocity, while forces and pressures are related to