This study used a custom-built horizontal atomic force microscope to compress individual chondrocytes under varying conditions in order to determine whether the cell's mechanical response is controlled by its cortex or internal mechanics. The researchers found that changing the compression rate, force, or pipette pressure that controls the cell's cortical tension drastically altered the chondrocyte's behavior. Specifically, tests where the pipette pressure was varied at a constant compression rate revealed that the cell's elastic response depended strongly on its cortical tension. This supports the cortical shell model of cellular mechanics for chondrocytes.

1. Single-Cell-Compression Experiments on Chondrocytes
using a Horizontal Atomic Force Microscope
Dean Pernas and Volkmar Heinrich
Department of Biomedical Engineering, University of California, Davis, 95616
Chondrocytes are responsible for the upkeep of healthy cartilage. Cartilage is found in joints
which are regions subject to a variety of strenuous forces and high pressures. Due to this,
chondrocytes must be able to resist high forces and shear stresses. Using single-cell
compression on chondrocytes, we wanted to find out whether the cell response was controlled
by the cortex, or by internal mechanics. Using a custom-built high-resolution horizontal
atomic force microscope, we compressed individual, micropipette-held chondrocytes under
varying cortical tensions and compression speeds. We applied compression forces up to
~1500 pN at indentation rates of 100 nm/s and 1 µm/s. These experiments were repeated at
pipette-aspiration pressures ranging from 2 cm to 12 cm of H2O. We found drastic changes in
chondrocyte behavior when any of the above factors were changed. The dependence of the
cell behavior on the compression rate exposed viscous components of the cell response. Other
tests performed at the same rate but different pressures revealed that the elastic cell response
depended strongly on the cortical tension.
Abstract
Aim of study
To examine the force responses of individual chondrocytes with high resolution under different
conditions.
a) Picture of acoustic enclosure housing the horizontal atomic force microscope
and vibrational platform. b) Atomic force microscope atop vibration-isolation
platform inside the acoustic enclosure. The water pressure device is also placed
on the vibration-isolation platform and is used to control the pipette pressure
acting on the chondrocyte. The aspiration pressure is calibrated to zero before
starting the experiment using the ambient pressure in the chamber.
Results
Example of a force cycle capturing the three phases involved in compressing the
chondrocyte. The first phase (downward slope) is the approach portion. It is
followed by the relaxation phase in which the chondrocyte is kept stationary to
monitor the cell’s relaxation. Finally the chondrocyte is retracted to its original
position. This entire cycle is repeated at least ten times.
Force-indentation curves of a chondrocyte aspirated at two different pipette
pressures. Time has been converted to the change in axial cell dimension during
the compression phase. For each pressure, two different cycles are shown,
demonstrating the excellent repeatability of this force experiment. At the lower
pressure, the chondrocyte is indented twice as much, revealing a clear
dependence of the cell stiffness on the aspiration pressure.
In this schematic, a traditional vertical AFM is oriented sideways. The laser is
reflected off the back of the cantilever. The reflected laser beam is detected by
the quadrant photodiode, which translates cantilever deflection into voltage
(optical lever method). Before each experiment, the cantilever spring constant is
calibrated, which allows us to relate cell indentations to compression forces.
Setup
Background
The mechanical behavior of cells usually falls under two different models, the cortical
shell model and the viscoelastic model. Our experiments were designed to examine
which model is more likely to govern the elastic response of chondrocytes.
Further translation of the pipette pushes
the chondrocyte against the cantilever. The
resulting cantilever deflection is recorded
using the optical lever method. The axial
dimension of the cell (D) is inferred from
the cantilever deflection and the known
displacement of the pipette.
Overview of the experimental setup. A
pipette-held chondrocyte is brought into
contact with the flat of an AFM cantilever.
The pipette is connected to a water
reservoir that can be moved vertically to
adjust the aspiration pressure with high
resolution. The initial cell dimension (D0) is
recorded (~7 µm in diameter).
Viscoelastic Model
This model dictates that the cell is divided up into two parts, an
cortical shell and a viscous liquid interior. The cortical tension
plays a key role in the elastic cell behavior.
This model predicts the mechanics of the cell as, essentially, a
spring. This implies that the entire cell is made of a solid-like
homogenous material which exhibits a linear response to force.
Acknowledgements
Christine Hastey, Jeni Lee, Gina McBarb, Andrew Morss
Cells exhibiting
this behavior:
-Red blood cells
-Neutrophils
Cells commonly
analyzed with
this model:
-Chondrocytes
Cortical Shell Model
Acknowledgements
Christine Hastey, Jeni Lee, Gina McBarb, Andrew Morss
Conclusions
Our results show that the pipette pressure acting on the chondrocyte has a direct impact on
the mechanical cell response. This indicates that the cortex of the chondrocyte dominates its
elastic response during small deformations. This behavior is consistent with the cortical shell
model.
Acknowledgements
We are grateful to Christine Hastey, Jeni Lee, Gina McBarb, and Andrew Morss for their
support of this project.
Key advantages of horizontal AFM
• Provides a unique side view of the test object and its interactions with the cantilever
• Coupling with micropipette makes the instrument extraordinarily versatile
• Cells are held at low suction pressure in a pipette, which eliminates the need to chemically
attach them to a carrier surface.
• Mechanical measurements can be performed anywhere along the length of the cantilever,
which provides a variable local cantilever spring constant and expands the range of applicable
forces.
• Mechanical tests can utilize the flat surface of the cantilever, which makes many experiments
(particularly cell-indentation tests) more amenable to rigorous quantitative analysis