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The need to measure the mechanical properties of skin,
scar, tumors, extracellular matrices, and wound tissue has been a goal of
researchers since the 1970s. A variety of methods have been used to evaluate
the mechanical properties of tissues over the last 40 years including uniaxial
and biaxial tensile testing, indentation and rotational tests, ultrasound
elastography (UE), optical cohesion tomography (OCT), optical cohesion
elastography (OCE), and vibrational analysis combined with OCT.
We have developed a test using vibrational OCT to
image and measure the moduli of the components of skin and scar tissue
non-invasively and non-destructively. Using images generated by OCT, maps of
the modulus as a function of position can be generated that will be useful in
marking the margins of scars, tumors as well as to evaluate the effects of
cosmetic treatments to the skin.
Key Words: Collagen,
Extracellular matrix, Optical coherence tomography, Vibrational analysis, Skin,
Scar, Tumor, Tumor margins
Measurement of the Modulus of Elastic Tissue and Collagen Fibers in Skin
and Decellularized Dermis
Since the properties of skin are time-dependent
and also depend on the rate of deformation of the sample, it is important to
separate the elastic properties from the viscous properties [3]. The model used
in most of our studies was decellularized dermis since the mechanical
properties of this material are similar to normal skin and decellularized
dermis can be freeze dried and stored at room temperature in a sterilized form
[3,5-8]. In addition it is also composed primarily of collagen fibers with some
elastic tissue still present simplifying interpretation of the mechanical
properties [3,5-8].
The non-linearity of the stress-strain curves
was dealt with by dividing the stress-strain curves into elastic and viscous
components [3,5-7, 9,10] (Figure 1). The elastic component was measured
as the stress at equilibrium in an incremental stress-strain experiment [3,10].
This test was conducted by the sequential addition of loading increments,
followed by a relaxation period between each loading step [3,10] (Figure 1). The elastic stress-strain curve was broken
into a linear low modulus region and a linear high modulus region [3, 5-7].
Equilibrium stress-strain curves were then used to calculate the elastic
modulus as the tangent to the curve which turned out to be about 0.5 MPa and 15
MPa, respectively for the low and high strain regions [3,5,10]. The low strain
region modulus was similar to that reported for elastic tissue and the high
strain modulus was similar to that reported for collagen fibers [7,10]. The
major problem with this approach was that the time required for the stress to
reach equilibrium in the skin and in decellularized dermis was as long as 24
hrs and the test resulted in destruction of the material.
The modulus of a number of ECMs was tested using this approach and the
results indicated that the elastic modulus was independent of strain-rate for
strain rates up to 10,000% per minute [10]. When the slope of the elastic
stress-strain curve at high strains is divided by the collagen content of the
tissue, the fraction of collagen aligned with the tensile direction, and the
change in strain after the collagen fibers of the tissue are stretched in
tension, the resultant modulus was calculated to be between 4.0 and 7.0 GPa,
values similar to those reported for the stiffness of the collagen molecule
[10,11]. The slope of the viscous stress-strain curve reflected the length of
the collagen fibrils and the viscous sliding of collagen fibrils by each other
during tensile deformation [10].
Results of theoretical modeling studies
suggested that the high strain elastic modulus was consistent with stretching
of the collagen triple helical regions devoid of proline and hydroxyproline
[12]. Therefore, the stiffness or modulus at high strains reflects the
resistance to stretching of the collagenous components of skin and is a useful
parameter to evaluate skin properties and their changes during wound healing
and disease processes. Results of studies on hypertrophic scar tissue have
shown that the major difference between scar and normal skin mechanical
properties lies in the inability of scar tissue to reorient under an applied
load in the same manner as does normal skin when stretched in tension [13].
However, since this approach required long time intervals and destruction of
the tissue it would not be considered useful in studying skin mechanics in vivo. Therefore, our efforts became
focused on development of a non-invasive and non-destructive test to measure
the mechanical properties of skin in vivo.
Development of Vibrational Optical Cohesion
Tomography (OCT)
The concept of development of a test to measure the
mechanical properties of skin came from the observation that when one vibrates
a bowl of soft material like jello with a hard inclusion, the jello vibrates at
a higher frequency than the inclusion. If one were able to measure the
frequency of vibration of the jello and inclusion separately then the frequency
of vibration of each material should be related to the material stiffness. It
turned out that a technique termed OCT created images of a substrate by
comparing a beam of light with part of the beam that is reflected off a
substrate [14]. If one measures the displacement of each substrate at varying
frequencies during vibration it is possible to calculate the modulus of each
the material. Figure 2 shows a plot of weighted displacement versus frequency
for decellularized dermis showing one resonant frequency (frequency of maximum
displacement) for collagen in decellularized human dermis.
Recently, we have reported the use of
vibrational analysis and OCT to characterize the mechanical behavior of
decellularized dermis, pig skin, bovine cartilage and subchondral bone [5,6].
Our results indicate that Poisson’s ratio for decellularized dermis ranges
between 0.38 and 0.63, values that are significantly different than 0.5 making
the assumption that Poisson’s ratio for skin and other collagenous tissues is
0.5 incorrect. This may lead to errors in calculating models dependent on an
exact value of this ratio [4]. In addition, a relationship was shown to exist
between the resonant frequency and the elastic modulus [5, 6]. The modulus
measured using vibrational OCT and that determined from tensile incremental
stress-strain curves for decellularized dermis and silicone rubber had a
correlation coefficient in excess of 0.95 demonstrating that the modulus
measured using vibrational OCT was very similar to the tensile modulus measured
using incremental stress-strain curves [5,6]. The relationship between the
modulus determined from vibrational and tensile testing is given by equation
(1) where Ev and Et are the moduli determined from vibrational and tensile
measurements in MPas.
Ev=1.026
Et + 0.0046 (1)
The measured value of the moduli did not
depend on an assumed value of Poisson’s ratio. Results of studies on
decellularized dermis and silicone rubber at frequencies of between 50 and 1000
Hz suggested that the viscous component of the modulus measured at frequencies
at or above the resonant frequency was 3%-4% [15]. Based on these results the
modulus measured using vibrational OCT was considered to be an approximation of
the “elastic modulus” and the viscous component was negligible [15]. While the
modulus determined from vibrational OCT is an approximation of the elastic
modulus it depends on the strain since not all the collagen fibers are recruited
to bear loads at low strains [16] (Figure 3).
Use of Vibrational OCT to Measure the Moduli
of Skin in Vivo
Vibrational OCT has been used to image and
measure the mechanical properties of skin and scar tissue in vivo. Results of in vivo
vibrational OCT studies suggest that the modulus of normal skin is lower than
that of scar tissue [16] and that of the margins of a healed scar have a
resonant frequency different than that of normal skin (Figure 4). Recent
results indicate that the margins of a scar can be mapped using vibrational OCT
since the resonant frequencies and moduli seen at the interface reflect both
the resonant frequency of normal skin and that of the scar tissue.
CONCLUSIONS
Using vibrational OCT the resonant frequency and
moduli of the components of skin and scar tissue can be measured non-invasively
and non-destructively. The numbers generated reflect to a first approximation
the elastic moduli and do not depend on measurement of other parameters. The
technique in vitro is calibrated
using incremental tensile measurements and vibrational OCT results on the same
sample. Using images generated by OCT, maps of the modulus as a function of
position can be generated that will be useful in marking the margins of scars,
tumors as well as to evaluate the effects of cosmetic treatments to the skin.
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