Mechanobiological effects on

cells in selected tissues

 How cells from selected tissues respond
biologically to mechanical forces?

We will limit our discussion to vascular tissue,

cartilage, and bone; these tissues are
particularly interesting to biomechanical
engineers because they are constantly
subjected to mechanical loading, and
mechanical factors are associated with
growth, maintenance, and pathology in these
tissues.
Endothelial cells in the vascular system
Endothelial cells line all blood-contacting surfaces
in the body
They have a number of functions, including
regulating the passage of water and solutes
across the vessel wall.
They are also sensitive transducers of the
mechanical forces imposed on them.
To make this more concrete, consider cells lining
a large artery.
These cells are subjected to a shear stress from
the passage of blood adjacent to their surfaces.
They are also subjected to a cyclic distension
from the pulsation of the artery during the
cardiac cycle.
 This combination of shear stress and stretch
affects the endothelium in several ways.

1. Endothelial cells rearrange their cytoskeleton and

change their shape in response to elevated levels
of shear stress (Fig.)
Specifically, the stress fibers of the actin
cytoskeleton at the base of the cell extend and
align with the direction of flow.
This occurs by regulated actin polymerization at
the ends of the fibers, which drives protrusion of
the cell membrane in the flow direction
This presumably acts to stabilize the cell and
improves resistance to shear forces
2. Arteries change their caliber in response to long-term
changes in blood flow brought about by alterations in
the distal vascular beds
This process, known as arterial remodeling, is a
physiological response that ensures that the artery is
the right caliber to deliver the required blood flow
rate
It is known that the endothelium is required for this
remodeling to take place
Further, a given artery will remodel itself so as to
maintain a target value of shear stress, equal to 15
dynes/cm2 for many (but not all) arteries.
Presumably this remodeling process is driven by the
endothelium sensing a local time-averaged shear stress
and then inducing the artery wall to remodel itself to
maintain a target value of this long-term shear stress.
3. In cell culture, vascular endothelial cells will
alter their production of many substances
(prostacyclins, nitric oxide, etc.) in response to
imposed shear stress
This effect can also be elicited by cyclic
stretch, and in fact these two stimuli seem to
be synergistic in some cases
Secretion of such molecules is almost certainly
how the endothelium signals the artery wall
to remodel in response to changes in time-
averaged shear stress
Smooth muscle cells in vascular tissue
Smooth muscle cells are found in several tissues in the
body including the genitourinary, respiratory, and
digestive tracts, and in blood vessels.
Vascular smooth muscle cells make up the cellular
component of the medial layer of blood vessels
The cells are aligned circumferentially around the
blood vessel, an orientation that allows them to control
the diameter of the vessel by contracting or relaxing.
Like endothelial cells lining a large artery, smooth
muscle cells are also subjected to cyclic stretching as
the artery distends during the cardiac cycle.
This stretching can affect smooth muscle cells in a
variety of ways.
1. Mills et al. used the biaxial stretch device to
cyclically stretch smooth muscle cells isolated
from the aortas of cows

They used the non-uniform strain profile to their

advantage in these experiments by seeding cells
only in the center or on the periphery of the
membrane to determine the effect of different
levels of strain magnitude
 The cells in the center of the membrane (where the
strains were 07%) grew more quickly than did smooth
muscle cells on the periphery of the membrane or cells
that were grown under static conditions.

The cells on the periphery of the membrane,which

experienced much higher strain levels (up to 24%
higher), also grew more quickly than cells grown
statically

Additionally, the cells on the periphery aligned

themselves circumferentially in response to the strain
stimulus, assuming an orientation similar to that
observed in blood vessels.
2. Cyclic stretch also increases the production of
extracellular matrix proteins (such as collagen
and elastin) and makes the smooth muscles
more contractile
 Kim et al. took advantage of the responsiveness of smooth
muscle cells to mechanical signals to engineer smooth muscle
tissue. They seeded vascular smooth muscle cells into three-
dimensional collagen sponges and stretched them
longitudinally at 7% strain (1 Hz) for several weeks

They found that the strain stimulus increased the proliferation

of the smooth muscle cells, increased the production of
collagen and elastin, and in the long term led to organization
of the tissue matrix.
Most importantly, the ultimate tensile strength and elastic
modulus of the resulting tissue were increased 12- and 34-
fold, respectively, over those for tissues grown without
mechanical stimulation.
 Chondrocytes in articular cartilage
Chondrocytes (cartilage cells) deform in their
matrix

This is important because articular cartilage is

subjected to a wide range of static and dynamic
loads, with peak stresses as high as 20 MPa and
compressive strains of up to 45%

Those loads can have profound effects not only on

the mechanical integrity of the tissue matrix but
also on the biological response of the
chondrocytes
 As with many cells, it is difficult to study the response of
chondrocytes to mechanical stimuli in vivo.
Instead, in vitro testing is performed and a platen
compression device is often used

The test samples are usually either small cartilage explants

(e.g., cylinders cored out from articular cartilage of the knee)
or synthetic systems comprising chondrocytes embedded in
three-dimensional hydrogels

These types of samples are used because they emulate the

native cellECM structure
 Chondrocytes respond very differently to compressive
forces, depending on the nature of the stimulus

1. Static compression of as little as 15% significantly

inhibits the synthesis of cartilage matrix components,
such as proteoglycans and proteins

After the static compression is released, biosynthetic

levels can return to normal,with the time to recovery
dependent on the duration and amplitude of the
original static compression
2. Dynamic (cyclic) compression can stimulate matrix
production considerably

However, the response to dynamic compression is very

dependent on the frequency and amplitude of the
compression.

For instance, low-amplitude compression (<5%) at a

lowfrequency (<0.001 Hz) had no effect on
proteoglycan or protein synthesis by chondrocytes in
articular cartilage implants from calf knee joints

At higher frequencies of stimulation (0.011 Hz),

however,low-amplitude compression resulted in up to
a 40% increase in proteoglycan and protein synthesis.
 The rate at which compressive strain is applied is also
an important parameter

For instance, Kurz et al. showed that a 50% strain

applied to cartilage explants at a rate of 0.01 s1 had no
effect on chondrocyte activity or matrix properties,
whereas higher rates of 0.11 s1 caused significant
decreases in proteoglycan and protein synthesis and
increased cell death

Interestingly, the cells that survived the injurious

compression of high strain rates were no longer able to
respond normally to low-amplitude cyclic compression
 On first glance, uniaxial compression seems like a
relatively simple loading condition

However, compression not only causes

deformation of the chondrocytes and matrix but
also leads to hydrostatic pressure gradients, fluid
flow, electrical streaming potentials (from
movement of ions), and other physicochemical
changes

Each of these mechanical, chemical, and electrical

stimuli likely plays some role in affecting the
biological response of chondrocytes.
 In an attempt to determine which stimuli are most
important, several researchers have developed
theoretical models to predict these various signals

By correlating spatial patterns of matrix synthesis

observed in cartilage explants during compression with
patterns of mechanical, chemical, or electrical stimuli
predicted by theoretical models, conclusions regarding
the most important stimuli can be made

Studies like these suggest that fluid flow within the

tissue and deformation of the ECM are critically
important at regulating ECM synthesis by chondrocytes
in response to dynamic compression
 In addition to experiencing compression, cartilage in
many joints is subjected to shear deformation

Devices similar to the compression device have been

built to apply shear forces to cartilage explants

This type of loading also affects chondrocyte

function; for instance, a dynamic shear deformation
of 1% at 0.1 Hz for 24 h increases proteoglycan and
protein synthesis by 41% and 25%, respectively
 Many of the studies on the effects of mechanical forces on cartilage
have been motivated by a desire to understand the role of
biomechanics in cartilage deterioration, such as occurs in
osteoarthritis

However, the understanding gained from these basic studies might

also have application for the repair and replacement of damaged
cartilage tissue

Several researchers are currently investigating how mechanical

stimulation might be used in the laboratory to grow or engineer
better replacement cartilage tissue

Initial studies with this approach indicate that significant

improvements in the mechanical properties and composition of
engineered cartilage can be obtained with dynamic compression

This mixture of biomechanics and the relatively new field of tissue

engineering is exciting and might be critical to obtaining
functional engineered tissues that are useful clinically.
 Osteoblasts and osteocytes in bone
Bone is an amazingly dynamic tissue that responds to
its mechanical environment

This adaptive behavior allows bones to withstand

repeated loading and alterations in loading without
breaking or sustaining extensive damage

The mechanisms by which bone senses and responds

to mechanical loads is an area of active research with
many unresolved questions

A review by Burr et al. summarized some of what is

known about bone adaptation to mechanical
stimulation, with focus on the tissue level responses
 Here we focus on in vitro studies of bone cells, with
particular attention to osteoblasts and osteocytes, two
types of bone cell that are believed to play important
roles in maintaining bone tissue

Osteoblasts are responsible for making new bone

Osteocytes are cells that were formerly osteoblasts but

have stopped synthesizing bone tissue and are now
embedded directly in the mineralized matrix of bone in
pores called lacunae

Osteocytes are not completely isolated from other

cells, however; they are connected to other osteocytes
and to osteoblasts on the surface of the bone matrix
through cell processes that travel through channels
(canaliculi) in the bone matrix
 As with cartilage, the loads applied to bones at the
tissue level are experienced by the cells residing in the
bone matrix

This type of mechanical stimulation (deformation of

the substrate to which the bone cells are attached) has
been emulated in vitro with a variety of devices,
including the uniaxial and biaxial stretch devices

The general conclusions from these studies are that

substrate deformations of approximately 110%
influence a variety of measures of bone cell function,
including DNA synthesis, enzyme production, synthesis
of collagen and non-collagenous proteins, and mineral
production.
 For instance, Ziros et al. found that mechanical
stimulation of human osteoblasts causes an increase
in the expression and DNA-binding activity of a
transcription factor known to be critical to osteoblast
differentiation
In this study, the osteoblasts were stretched with an
average strain of 2.5% using a device
 It turns out, however, that because of the relative
stiffness of mineralized bone tissue, peak strains on
the surface of most bones do not usually exceed
0.3%

It is possible that in certain circumstances matrix

deformations might exceed 0.5%, for example, in
pre-mineralized bone tissue during fracture repair or
in regions where the microstructure of the bone
results in local strain concentrations

In general, however, the physiological relevance of

the in vitro studies that apply strains in excess of 1%
is unclear
 More recent studies have focussed on the response of bone
cells to fluid flow rather than substrate deformation.
Mechanical loads applied to bones not only deform the
matrix, but also cause movement of extracellular fluid
through the lacunae and canaliculi in the bone matrix

This movement of fluid is hypothesized to stimulate

osteocytes and osteoblasts through fluid shear stress
effects, streaming potentials, or chemical transport
mechanisms

Osteocytes, owing to their location with the lacunae, may

therefore act as mechanosensors, sensing fluid flows and
signaling to osteoblasts (through their cell processes, for
instance) to cause alterations in bone formation.

Consistent with this theory, bone cells are more sensitive to

physiological levels of fluid flow than they are to
physiological levels of stretching, as shown in two studies.
1. Smalt et al. measured the biological response of a
variety of types of osteoblast to stretch and shear
stress by monitoring the production of nitric oxide and
prostaglandins, two cellular products known to be
mechanically responsive in vivo

Using the stretch device , they showed that the

osteoblasts did not respond to uniaxial strains of 0.05
0.5% but did produce nitric oxide and prostaglandins
when subjected to a period of steady shear stress of 148
dynes/cm2 (applied using a parallel plate flow chamber)
2. You et al. reached the same conclusion but used more
physiologically relevant fluid flows

Theoretical models of fluid flow in the lacunar

canalicular network predict the flow will be oscillatory
(i.e., reversing), with wall shear stress of the order of
830 dynes/cm2

Uniaxial stretching of 0.5% strain at 1 Hz had no effect

on the expression of osteopontin mRNA by the bone
cells. (Osteopontin is a non-collagenous protein
important in bone formation and shown to be
modulated by mechanical stimulation.)

When they subjected the cells to oscillatory (1 Hz) fluid

flow in a parallel plate flow chamber, with shear
stresses of 20 dynes/cm2, osteopontin mRNA
expression went up significantly.
 As with cartilage biomechanical research, the
motivating factors for understanding how
bone responds to its mechanical environment
are bone diseases and failures.

And as with cartilage, recent developments in

tissue engineering have spurred interest in
using mechanical stimulation to engineer
replacement bone tissue

Initial studies have shown promising results.

 Using three-dimensional titanium mesh matrices
seeded with osteoblast precursor cells, Bancroft et
al. showed that mineralized bone tissue formed
more rapidly in matrices perfused with cell culture
media than in matrices not subjected to fluid flow

Interestingly, this effect occurred with flow rates as

low as 0.3 ml/min (corresponding to an estimated
shear stress of 1 dyne/cm2)

Although it is not clear from these experiments how

a stimulus of such low magnitude could cause an
increase in bone formation, the results do suggest
that mechanical stimulation will be valuable for bone
tissue engineering.
Models of cellular
biomechanical
behavior
 Experimental data are clearly necessary for
characterizing the mechanical behavior of cells
However, it is often desirable (and sometimes
even essential) to develop models of the
mechanical behavior of cells
Such mechanical models help us to understand
and interpret experimental observations
They are also useful for predicting the response
of a cell to a variety of inputs and for comparing
responses of different cells
 The first model considers the cell as a viscoelastic body
essentially made up of viscous protoplasm surrounded by a
cell membrane

This approach considers the mechanics of the whole cell,

without considering the contribution of individual cellular
components, such as the cytoskeleton

The second model focuses on how the mechanical

properties of a cell are determined by the structure and
composition of its cytoskeleton. Each of the first two
models considers the behavior of the cell in isolation

The third model

In reality, cells reside within an extracellular matrix and the
properties of the matrix and its interaction with the cell will
affect the mechanical behavior of the cell in response to
force applied to a tissue.
Lumped parameter viscoelastic model
of the cell
 The data from the magnetic bead rheometry
experiments indicate that the cells
response to a step application of force is
viscoelastic, with an immediate elastic
response (phase 1)followed by gradual creep
(phases II and III).
The viscoelastic behavior of materials
(including cells and many biological tissues)
can be modeled using lumped parameter
models constructed from arrangements of
two simple elements: the linear spring and the
dashpot.
 we construct a viscoelastic model of the cell and compare its
predictions with those obtained experimentally
It is important to understand that the elements in these
models cannot be unambiguously associated with specific
cellular components; instead, we represent the action of all
elastic components of the cell by a linear spring (or springs)
and the action of all viscous components by a dashpot (or
dashpots).
Maxwell body
Kelvin body
Bausch model
 This lumping of the response of a complex
biomechanical system into a small number of
elements is obviously a simplification.
Nonetheless, such lumped parameter models
are easy to work with and represent a good
first approach to quantifying cellular
biomechanics.