Design of an In-Vivo Probe to Detect Cartilage
Degeneration
by
Emerson Cheung Quan
Submitted to the Department of Aeronautics and Astronautics
in partial fulfillment of the requirements for the degree of
Master of Science
at the
MASSACHUSETTS INSTITUTE OF TECHNOLOGY
June 1998
@ Massachusetts Institute of Technology 1998. All rights reserved.
Author .............
Department of Aeronautics and Astronautics
May 22, 1998
Certified by......
/
. . .
Alan J. Grodzinsky
Professor
Department of EECS, Mechanical, and Bioengineering
Thesis Supervisor
Accepted by ..............
\
Jaime Peraire
Associate Professor
Chairman, Department Graduate Committee
i~L tbJA~9
Design of an In-Vivo Probe to Detect Cartilage
Degeneration
by
Emerson Cheung Quan
Submitted to the Department of Aeronautics and Astronautics
on May 22, 1998, in partial fulfillment of the
requirements for the degree of
Master of Science
Abstract
Currently, detection of the onset of osteoarthritis is very difficult without using destructive techniques. Without early detection, pharmaceutical intervention to reverse
or prevent osteoarthritis cannot be achieved. A technique developed by Frank and
Grodzinsky takes advantage of an electromechanical property inherent in cartilage.
Upon the introduction of an electrical field to the cartilage surface, a mechanical
stress at the surface is produced via an electrokinetic effect. This electrokinetic effect
in cartilage is a function of the molecular composition and integrity of the tissue.
Past designs to measure this phenomenon have now resulted in a handheld in-vivo
probe that utilizes the technology of piezo-electric films. These piezo-electric films
can sensitively measure the mechanical stresses developed within the cartilage and
convert them into a quantitative electrical signal. Potential applications of this probe
include use in arthroscopic surgery to provide surgeons with more quantitative information for diagnostics and therapeutic intervention. By testing the probe on human
tissue and validating such measurements, the goal of this thesis is to show that the
size of the handheld in-vivo probe can be reduced for arthroscopic surgery and that
the measurements made by the probe can be correlated to the physical properties of
cartilage.
Thesis Supervisor: Alan J. Grodzinsky
Title: Professor
Dept: Department of EECS, Mechanical, and Bioengineering
Acknowledgments
It's amazing how two years can go by so quickly. In the past two years, I've been
very fortunate to have spent that time working with a bunch of wonderful people.
First of all I must acknowledge the very man who leads us, his troops, in the lab. Al,
the Godfather of Cartilage, who has taught me the blend of good research and good
nature. What more can I say besides the fact that I've never seen anyone consume an
entire rain forest of coffee beans in a year like Al. We practically run a Starbucks in
the back of our lab. I'd like to thank the rest of the supporting cast for all of their help
(I could write an entire thesis about how great each of these people are, but for the
sake of our reviewers I'll keep it short and sweet). First to my crew - the probesters.
What a fitting name to a bunch of guys who know all about probes. To Steve "Can I
take a swig of that?" Treppo, you've been the best labmate anyone could ever have.
I am always deeply in debt for your advise and generosity in helping me around the
lab. To David Breslau, who's amazing craftsmanship, helped manufacture the parts
of the probe. To Jeff, my partner in crime, thanks for the tag team effort on the
project. And of course Nik, who has convinced me that camping out in the middle of
winter isn't just a spectator sport. Around the lab I'd like to thank some others. To
Linda who's mix of crass humor and kind heartedness always brought a smile to my
face. And who can forget Han Hwa, our lab mom. Without her I probably would've
accidently blown up the lab by now. Then there's Eliot, who's ability to fix anything,
has never ceased to amaze me. And finally there's the rest of the gang: Paula, Marc,
Andy, Niti, and Vicki. Thanks for all the fun times and the constant reminder that
grad school is all about Tosci's and procrastination.
I'd also like to thank people in the Aero/Astro department. To Dava, a fellow
Domer, who has continually inspired me in the field of space physiology, the MVLers,
Liz Zotos who has helped me in all aspects of my graduate career, and finally the
NASA Space Grant Consortium for keeping the tuition bill out of my hands and
keeping a roof over my head.
I'd also like to thank all my friends at Ashdown. Especially my roommates
for putting up with me and my stresses. Tom, Frank, and Ben - you guys are the
greatest. I'd also like to give a "shout out" to others around the asylum. To Amy,
Emily, and Priscilla, thanks for being there through the good times and the bad. And
also to my friends at home, Larry, Derek, Bim, Mar, and Sherwin - thanks always for
your constant support. And finally I'd like to thank my parents for all their years of
hard work, love and support. This day would not have been possible without them.
Emerson C Quan
May 22, 1998
To Andres and Margaret
with love
Contents
13
1 Introduction
1.1
History .................
1.2
Epidemiology ...................
1.3
Pathogenesis ...................
1.4
Diagnosis
...................
1.5
Treatment ...................
1.6
Overview ..................
..
....
..
........
..
13
..
14
..
14
...........
..
15
...........
..
16
.........
..........
17
.....
.........
18
2 Background and Theory
2.1
Cartilage ...........................
2.2
Composition . ..................
2.3
2.4
..
2.2.2
Proteoglycans ................
2.2.3
Chondrocytes .....
.
22
.
. . .
22
...
.
23
..
Cartilage Electromechanics . . . . . . . .............
Streaming Potential .........
2.3.2
Current Generated Stress
19
.......
..................
2.3.1
19
..
.........
Collagen ...................
19
...
........
2.2.1
18
..
....
.
...........
23
....
................
.
Previous Research on Surface Spectroscopy . ............
31
3 Design and Manufacturing
3.1
Probe Construction ...................
3.1.1
Inner Core ...................
3.1.2
Insulating Sheath ...................
24
.......
........
...
...
.
32
.
32
33
3.1.3
3.2
Outer Stainless Steel Body . . . . . . . . . . . .
Fabrication of ETS .........
.. .... .... .
..........
3.2.1
Assembly
3.2.2
Photofabrication
3.2.3
Etching
3.2.4
Cutting and Mounting .
3.2.5
Fabrication of Silver/Silver Chloride Electrodes
......
...........
. ..... ... ..
.. .... ... ..
.. .... .... .
.. .... .... .
44
4 Experimental Method
4.1
4.2
4.3
Calibration
. .
.............
4.1.1
Hardware setup . . . . . . .
4.1.2
Procedure . .........
Current Generated Stress Experimen
. . . . .
45
. .
.
45
. .
.
46
Hardware Setup . . . . . . .
. .
.
46
4.2.2
Testing for Parasitic Signals
. .
.
46
4.2.3
Tissue Experiments with the
. .
.
47
. .
.
47
. . . . .
47
Confined Compression
. . . . . ..
4.3.1
Hardware Setup . . . . . . .
4.3.2
Procedure . .........
............
Biochemistry
4.5
Version 5.0 Validation
. . . . . .
. . . . . . ...
4.5.1
Calibration
4.5.2
Procedure . .........
5 Results
5.2
45
4.2.1
4.4
5.1
.
Patella Experiments
5.1.1
Correlations ............................
5.1.2
Joint Comparisons
5.1.3
Comparison of Articulating Cartilage Surfaces .........
........................
Version 5.0 Results ............................
. .
.
49
. .
.
49
. .
.
51
. . . . .
51
. . .
..
51
.
6 Conclusions
72
6.1
Significance of results ...................
6.2
Issues
6.3
Looking Ahead ...................
...................
....
............
........
. .
.
72
..
73
..
73
A Version 4.0 Probe
75
B Version 5.0 Probe
78
B.1 First Iteration of ETS Patterns
...................
........
B.2 Second Iteration ...................
8
..
81
..
82
List of Figures
2-1
Collagen, proteoglycans, and chondrocytes are linked together to form
. ..
Extracellular Matrix (ECM). [Courtesy of S Berkenblit] .....
20
21
.........
2-2
Structure of proteoglycan. [Courtesy of S Berkenblit]
2-3
Current Generated Stress (CGS). The negative fixed charges on the
ECM move toward the positive electrode while the ions in the fluid
move toward the negative electrode. By driving a sinusoidal current,
the ensuing motions of the fluid and solid phases create a mechanical
.
stress within the cartilage that can be measured at the surface....
2-4
Cartilage is uniaxially confined between two silver electrodes to pro. .
..
duce current generated stress. ..................
2-5
26
Results of a theoretical model showing the flow of the current density
27
coming from two electrodes on the same side of the testing surface.
2-6
25
A variable wavelength in-vitro probe. Different polarity configuration
give different wavelengths and penetrate at various depths of the tissue.
[Courtesy of S Berkenblit]
2-7
....
.........
.......
An experimental setup to test the handheld probe on a disc of cartilage.
...
[Courtesy of D Bombard] ...................
3-1
.
29
.
32
.
34
Diagram of the sheath placed over the ETS and head of the inner core.
35
..
Diagram of the inner core. A - Hollow stainless steel tube, B - Torlon
body ...................
3-3
..
Three parts of the version 5.0 probe. A - Inner core, B - Insulating
sheath, C - Outer stainless steel body ................
3-2
28
. ....
................
3-4
Diagram of the outer body covering the probe's interior. A nut screws
. ....
.......
the probe parts tightly together .........
3-6
Patterns for the electrodes.
.........
39
....
41
.. ..
Left - Crucifix backing plate attached to kynar side of ETS, Right 42
...................
Schematic of proposed measurements in an joint cavity, Right - Picture
....
of an assembled v5.0 probe. .............
4-1
. . . .
..
..........
Silver electrodes after chloridation.
3-9
............
After etching and cutting the ETS. Left - Kynar electrodes, Right Silver electrodes .
3-8
38
Left - Piezo electrodes, Right - Silver
electrodes. [Courtesy of S Treppo] ....
3-7
. .... .
..
3-5 Dimensions of each layer of ETS materials. . ........
36
..
43
...
Setup for an intact joint experiment. Patella is placed in a holder and
submerged in testing solution. Probe is inserted into a probe sheath
clamped to a ring stand. The end of the probe is placed on a flat surface
of the joint. A calibrated spring in the shaft provides the required static
offset stress........................
4-2
. ...
.. ..
.
48
.
Test sites were drilled and a 9.5 mm diameter core was extracted from
the joint. A 1.0 mm thick slice of the cartilage surface was microtomed
from the core. From each slice, four 3.0 mm discs were punched out.
One disc was tested in confined compression while the others were
4-3
50
.......
tested for biochemistry. ...................
Polarity configurations for A - short and B - extra long wavelengths.
O in electrodes 2 and 3 for (B) denote no current output. The longer
wavelength in (B) penetrates deeper into the cartilage tissue. . ... ..
53
5-1
Calibration outputs from the left and right channels of a v4.0 probe. .
55
5-2
Current generated stress versus frequency, N = 8. . .......
5-3
Correlations of the current generated stress with A - equilibrium modulus, B - age, C - permeability, and D - water content.
. ......
56
...
.
58
5-4
Correlations of the electromechanical coupling coefficient with A streaming potential and B - dynamic stiffness. . ........
5-5
59
....
Correlations of age with A - equilibrium modulus, B - water content,
and C - GAG per tissue wet weight, also D - correlation of equilibrium
60
modulus to hydroxyproline content. ....................
5-6
Comparing electromechanical results of cartilage from various joint surfaces. Cartilage came from TA - talar, F - distal femur, TP - tibial
plateau, and PT - patellar joint surfaces. ....
5-7
62
..
..........
Comparing biochemical properties of cartilage from various joint surfaces. Cartilage came from TA - talar, F - distal femur, TP - tibial
plateau, and PT - patellar joint surfaces . .......
5-8
......
63
. ...
Comparing electromechanical results of cartilage from surfaces on the
patella that are in contact with cartilage from surfaces on the distal
femur. Comparison of data from the same human subjects are denoted
5-9
64
..
........
by FP-C and PT-C ..................
Comparing biochemical composition results of cartilage from surfaces
on the patella that are in contact with cartilage from surfaces on the
distal femur. Comparison of data from the same human subjects are
......
denoted by FP-C and PT-C .........
. ....
65
.....
5-10 Piezo response for each channel at various dynamic amplitudes and at
...
various frequencies ..................
.
. .
66
5-11 Differential amplitude and phase from channels 1-2 and channels 3-4
at various applied current densities and frequencies. Driving current is
in a short wavelength configuration. ................
67
...
5-12 CGS output from A - channels 1-2 and B - channels 3-4 at various
current densities and frequencies.
.
68
. .
69
...................
5-13 A - Differential amplitude from channels 1-4 at various applied current
densities and frequencies. Driving current is in an extra long wavelength configuration. B - CGS response from an extra long wavelength
configuration. C - Phase output. .................
..
5-14 Normalized stress amplitude for a short wavelength configuration in A
- channels 1-2 and B - channels 3-4. C - Normalized stress amplitude
for an extra long wavelength configuration in channels 1-4. . ......
70
5-15 Parasitic response from differential outputs and phase from channels
71
...
.....
1-2 and channels 3-4. ...................
5-16 Parasitic CGS from A - channels 1-2 and B - channels 3-4. ......
.
71
A-1 Parts of the v4.0 probe being assembled. Outer body diameter of the
probe is 1.0 cm. [Courtesy of D Bombard]
76
. ..............
A-2 Current v4.0 ETS electrode pattern. Left - Piezo electrode; Right Silver electrode. Cross patterns are used to align ETS wafers during
B-1 Dimensions of Inner core of the v5.0 probe. ....
. .
....
photofabrication. [Courtesy of S Treppo] . ......
....
. ..
...
...
.
77
79
B-2 Dimensions of A - Bottom part of inner core, B - Insulating sheath,
and C - Outer Body of the v5.0 probe .........
....
..
...
80
B-3 First iteration of v5.0 ETS electrode patterns. Left - Piezo electrodes;
Right - Silver electrodes [Courtesy of S Treppo]
81
. ...........
B-4 Second iteration of v5.0 ETS electrode patterns. Left - Piezo electrodes; Right - Silver electrodes [Courtesy of S Treppo] ....
....
82
Chapter 1
Introduction
Osteoarthritis (OA) is the most common disease that affects the everyday mobility and activities of an individual. The disease occurs in the tissue of synovial joints
(e.g., knees, hips, and hands) and is characterized by pain, tenderness, and limitations
in limb movement. Individuals afflicted with OA often experience a decrease in the
quality of life due to prolong discomfort and reduction in daily activities. Although
the disease itself is not a cause of death, it is the greatest cause of physical suffering
to those afflicted.
It is estimated that in the US alone, the number of people suffering from OA
will reach 68 million by the year 2010 [30]. The cost of treating OA is also staggering
with an 1989 estimate of 54.6 billion dollars alone in the US [47]. This amount is even
greater when considering the amount of work time lost by those afflicted. Research
in OA has yielded important knowledge about the disease and some of its causes.
Further research will be needed in order to discover viable ways to treat OA.
1.1
History
Osteoarthritis was prevalent even before humans existed, from evidence in the
fossils of ancient mammalian species [16].
The symptoms of the disease were well
known to ancient man, with the similar feelings of pain and discomfort experienced
today. Even by the early 1700s, the exact cause of OA was still a mystery, although
some such as English physician William Herberden began carefully documenting the
observations of lesions in their patient's joint tissue. By the mid-20th century, scientists had been able to separate the different causes of joint pain [16]. Among the
different subsets of joint disease include hypertrophic (degenerative joint disease), atrophic (rheumatoid arthritis), and OA. Rheumatoid arthritis (RA), which is different
in pathology from OA, is caused by inflammation in the joint cavity as a result of
some pathogen. The initial symptoms of RA are hard to distinguish from OA [38].
1.2
Epidemiology
Because osteoarthritis is non-discriminate in race, gender, and geography, epi-
demiological studies have helped classify groups predisposed to OA. OA is most prevalent in age groups above 65 (over 70% experience symptoms) with a higher incidence
among women than men over 50 [15]. The prevalence of OA generally increases with
age. Degeneration of the joint tissue can be seen in individuals as young as 20 years
with some abnormalities seen in all by the age of 40 [40]. In addition, individuals
having undergone major joint surgeries or experiencing inflammatory joint diseases
are more likely to experience OA. Other risk factors for OA include obesity, joint
injury, or a genetic predisposition for OA [30]. Interestingly, OA occurs in certain
joints at a much higher rate than others. For instance, OA is often seen in knees
and hips, but rarely in ankles or elbows [26]. Proper analysis of epidemiological data
must include both biological and mechanical factors which can cause OA.
1.3
Pathogenesis
The pathways of OA progression can be observed through the examination of
tissue. OA is seen to occur via two major pathways. The first is a breakdown of
the cartilage structure which leads to an erosion of the cartilage surface [39]. The
initial phases may be confined focally but eventually diffuses to other areas on the
surface. Mechanical stresses may induce additional pitting and fissures creating a
roughened surface containing ulcerations. Eventually deterioration takes place down
to the bone, eliminating the load bearing capabilities in the joint [40].
A second pathway occurs by the proliferation of cartilage and bony material at
the joint periphery [1]. This overgrowth of tissue leads to osteophyte formation and
results in incompatible congruity between joint surfaces.
But what events stimulate the initiation of OA? There has been much speculation that there is both a mechanical and biochemical interaction that initiates the
disease process. Some possibilities include the release of proteolytic or collagenolytic
enzymes from cartilage cells in response to mechanical stimuli which causes unraveling of matrix integrity [40].
Others believe there is a chemical or immunological
abnormality that elicits degradation [8]. Another possibility is that the balance between tissue repair and tissue degeneration is disrupted by either of the two pathways
above [10].
By studying the physiology of the tissue from the molecular level to the organ
level, OA progression can be monitored in order to understand the process of this
disease. Animal models can be used to simulate the progression of OA and as subjects
for new treatments. One example of a model for OA progression was created by
transecting the anterior cruciate ligament of dogs [46]. This enables an immediate,
observable progression to OA. By understanding the pathways of disease progression,
researchers can pinpoint areas to target for therapy.
1.4
Diagnosis
Efforts are being made to diagnose the disease progression at it's earliest stages,
in order to determine a treatment before further damage can occur. Initial diagnosis
of OA begin with patients complaining of pain and stiffness in their joints. Further
diagnosis can be made using the current gold standard of radiography [30]. In addition
a visual inspection can be made during surgery via an arthroscope. A grading scheme
to characterize the damage is utilized in both methods. The most commonly used
scale of radiographic evidence is the Kellgran and Lawrence method [27]. The scale
is numbered from 0 to 4 with 0 being no visible defects and 4 showing visible OA.
The Collins scale [9], on the other hand, is used during a surgical visual inspection
with the same definitions for a scale of 0 to 4. Both the Collins scale as well as
radiography fall short in their abilities to detect early onset of osteoarthritis. Visual
inspection can only confirm whether tissue has already become osteoarthritic. Visual
inspections cannot detect the precursors of OA in tissue.
Laboratory methods such as a synovial fluid extraction or a histological examination (Mankin scale) can be used to further investigate the progression of disease
[33]. Unfortunately, synovial fluid extraction can only rule out other possible causes
for the pain, such as RA. The Mankin scale categorizes the extent of disease progression in tissue via histology but requires a destructive biopsy. Furthermore, histological
examination occurs only where tissue was removed.
Other possible methods include MRI [45] and sonography [44], but they too have
limitations in detecting changes in cartilage. New techniques that nondestructively
test for OA and its precursors are the driving interests for this study.
1.5
Treatment
Currently there are no specific treatments that have shown the ability to cure,
retard or prevent OA in humans. Anti-inflammatory or analgesic medications can
typically be prescribed to give temporary relief from pain. In addition, a regimen of
rest and a change in one's pattern of physical activity can prevent worsening of pain.
There is much research being done in looking for long term solutions to the
problems of OA. Much research is going into the development of medical techniques
and drugs that could reverse OA. However, assessment of the treatment's progress
must be made in order to evaluate efficacy of the treatment. Therefore, innovative
diagnostic tools, beyond the visual methods that were previously discussed, must be
discovered to give a more accurate assessment of tissue integrity.
Prevention through early detection of OA is an important goal if at some point
reversal of damaged tissue is impossible. Therefore the discovery of drugs that can
target the onset of OA would be of great value to those at high risk for OA.
1.6
Overview
This thesis begins with a short overview of cartilage physiology. We will begin
by describing the electromechanical properties of cartilage followed by a discussion of
the theories of electrokinetic spectroscopy. The design of an electromechanical surface
spectroscopy device to probe cartilage is then described along with the experimental
protocol for calibration and subsequent testing of both bovine and human tissue.
Finally the last two chapters are devoted to the presentation and analysis of the data
along with some concluding thoughts on the results and future directions for our
device.
Chapter 2
Background and Theory
This chapter will review some of the basic properties of cartilage tissue and
its physiology. Understanding that cartilage is a dynamic tissue which reacts and
responds to external forces will enable the reader to appreciate the intricacies that
exist. It is important to learn how a biomedical device can take advantage of the
natural phenomenons that are a consequence of the properties of cartilage. The design
of the device is presented along with some introductory remarks on electromechanical
theory in regards to living entities such as tissue. And finally, previous research
leading up to the current discoveries will be summarized to give some perspective on
the progress that has been made so far.
2.1
Cartilage
Cartilage is an aneural, avascular, and living tissue that lines the ends of load
bearing joints. It macroscopically looks pearly white and homogenous yet is microscopically composed of a dense network of molecular structures. Cartilage protects
joint areas by spreading the compressive loading over a larger area, acting as a damping element during high impact loading. Cartilage is also strong in tensile loading, for
example during stretching of a spinal disk. Cartilage also enables frictionless movement between joints through the lubrication from synovial fluid in the joint cavity.
Lubrication protects the cartilage from trauma that could occur from shear stresses.
These mechanical properties of cartilage are a consequence of the composition and
unique physical properties it possesses.
2.2
Composition
Cartilage is comprised of mostly water (60 - 80% of total weight) and extracel-
lular matrix (ECM) [34]. The ECM is made up of collagen fibrils (types II,IX,XI),
charged proteoglycans (PGs), and cells (called chondrocytes). Collagen and proteoglycans form the framework for cartilage (Figure 2-1).
2.2.1
Collagen
Collagen makes up the majority of the dry weight in cartilage (approximately
50%) [32]. In cartilage the most abundant form of collagen is type II which acts
as the structural scaffold. Other types of collagen exist, for example type IX helps
connect the various matrix elements together while type XI regulates the size of fiber
formation [7]. Type II collagen is composed of three tightly interwoven alpha chains,
each with a repeating amino acid sequence of Gly-Pro-(Hydroxylysine) [52].
The
triple helical structure enables collagen II to have a high tensile strength. The highly
enriched amount of hydroxylysine helps collagen II link together the ECM network.
2.2.2
Proteoglycans
Proteoglycans themselves are attached to a core protein like hyaluronate via
a link protein. The number of linked PGs depend on the functional nature of the
cartilage [21, 23]. Some common PGs that can be found in cartilage include aggrecan,
decorin, and versican.
Proteoglycans are a protein chain that contain carbohydrate domains. Their
function is to help form molecular aggregates in cartilage [37]. Together PGs form
a hydrogel like structure that is immersed in collagen. This material helps cartilage
withstand the swelling forces that occur during compression. Attached to each PG
GAG chain
"
,I 11111'I',
eI t \
PG :
Vr
;;!i
a
"r
:
I
4I
Chondrocyte"
"
Figure 2-1: Collagen, proteoglycans, and chondrocytes are linked together to form
Extracellular Matrix (ECM). [Courtesy of S Berkenblit]
is at least one sulfated glycosaminoglycan (GAG) (Figure 2-2). Most PGs contain
two types of GAGs [13]. For example aggrecan is the major proteoglycan in articular
cartilage (around 90%) and has attached to it two types of GAGs - chondroitin
sulfate and keratin sulfate. The amount of GAG attached to each proteoglycan varies
depending on the functionality and integrity of the tissue [24].
oligo-
Hyaluronate
O-linked oligosaccharide
Link protein
Hyaluronate
IIII
KS-rich region
CS = Chondroitin sulfate
KS = Keratan sulfate
Figure 2-2: Structure of proteoglycan. [Courtesy of S Berkenblit]
GAGs are chains of repeating disaccharide units that contain highly charged
carboxylate and sulfate groups [36].
The high density of negatively fixed charge
groups helps attract ions of the opposite charge. These ions osmotically attract water
to help maintain a constant concentration and pH within the tissue [23, 35].
GAG chains attach to certain areas along the proteoglycan. In aggrecan, chondroitin sulfate (10 - 15kDa) attachment begins at all Ser-Gly dipeptide sites along
the proteoglycan [24]. The exact site of keratin sulfate (5 - 10kDa) attachment is unknown except for the fact that the majority of keratin sulfate is located in a proline
rich region behind the group of chondroitin sulfate [21]. There are approximately 100
- 150 GAG chains per aggrecan unit. Some other types of GAGs found in collagen
include heparin sulfate and dermatin sulfate.
2.2.3
Chondrocytes
Because cartilage is avascular, chondrocyte cells must acquire their nutrition
through the diffusion of molecules through the ECM matrix. These molecules come
from the synovial fluid in contact with the cartilage surface. Chondrocytes help maintain and repair the ECM structure by synthesizing new collagen and proteoglycans
[25]. In addition chondrocytes can adapt to the changes in mechanical loading through
their cell receptors [29]. These adaptations, done by balancing the homeostasis of the
matrix, maintain cartilage integrity.
2.3
Cartilage Electromechanics
Because of the composition in cartilage, certain properties are exhibited by
this tissue. Cartilage is a solid material, of which certain engineering properties can
be measured, most specifically stress, strain, and engineering stiffness. Dashefsky
once devised an indenting device that could measure the softness of the tissue in
order to assess areas that might be damaged [12]. Also because cartilage is a porous
material, the permeability of fluid through the matrix can be measured or estimated
by models. One model, known as the poroelastic theory, treats cartilage as a fluidsaturated porous medium whose viscous effects occur through the friction between
the fluid and solid materials [4, 5]. Another method, known as the biphasic theory,
separates the material properties and constitutive relations based on the phase of the
material [42]. Through these models fluid flow can be related to mechanical properties
like stiffness [41, 43]. Tests like uniaxial confined compression are used to measure
these mechanical properties.
Besides having purely mechanical properties, cartilage exhibits electrical properties that are coupled with mechanical stresses [22]. This electromechanical trans-
duction effect is a property of the cartilage composition, specifically the proteoglycans
[18, 31]. Two phenomenons are observed - streaming potential and current generated
stress [18, 19]. These properties can be modeled by combining electrokinetic coupling
laws with the poroelastic or biphasic theory [42].
Experimental tests have shown
results that corresponded well with theory.
2.3.1
Streaming Potential
Because cartilage has a high density of charged molecules, the tissue exhibits an
electrokinetic effect [18, 19]. Studies have shown that when cartilage is mechanically
compressed, the positive charged ions in the tissue fluid are convected away from
the negative fixed charges of the ECM [17].
This separation of charge creates an
electric field in the direction of the fluid flow called a streaming potential [20]. An
experimental protocol was devised which could measure the streaming potential with
a mechanical test like confined compression. This protocol is described in Chapter 4.
2.3.2
Current Generated Stress
As with the streaming potential, it has been proven that the reverse effect can
also occur. This effect, known as current generated stress, occurs when an electric
current is applied to the cartilage [18, 19]. The negative fixed charges of the ECM
move toward the positive electrode (by electrophoretic migration) while the ions in
the fluid phase move toward the negative electrode (by electroosmosis). By driving
a sinusoidal current, the ensuing motions of the fluid and solid phases create a mechanical stress within the cartilage (Figure 2-3). This stress can be measured at the
surface.
By measuring the current generated stress in cartilage, certain parameters can
be calculated using mathematical models that relate the current generated stress to
tissue material properties [49]. The altered state of the cartilage matrix caused by
degradation and loss of proteoglycan will change the current generated stress response.
Therefore the difference between normal and degraded tissue can thus be detected.
2.4
Previous Research on Surface Spectroscopy
One dimensional tests to measure the mechanical stress in cartilage produced
by an applied electric potential were first performed by Frank and Grodzinsky (1987).
They showed that by placing an excised disk of bovine cartilage between two silver
chloride electrodes and then applying a current, a mechanical stress was produced
[18, 19]. Their setup is shown in Figure 2-4.
Sachs and Grodzinsky (1989) later completed a mathematical model showing
that two silver electrodes placed on the same surface side of cartilage could induce
a mechanical response when current is applied [50, 49] (Figure 2-5). This technique,
called electromechanical surface spectroscopy, would measure the electromechanical
properties of joint surfaces in a nondestructive manner.
Berkenblit and Grodzinsky (1995) followed with experiments involving multiple
interdigitated electrodes to localize cartilage degradation to depths below the articulating surface [2, 3]. By designating different patterns of electrode polarity, wavelengths of various sizes could be created (Figure 2-6). The longer the wavelength,
the deeper the current density penetrated into the cartilage. By calculating the ratio
between measurements made from short and long wavelengths, a normalized measure
of surface versus full-depth tissue integrity could be found.
Bombard (1995) designed and constructed a prototypical handheld measuring
device to measure for current generated stress [6]. A two electrode probe of 1.0 cm
diameter was manufactured. On the end, the probe had an electrode transducer system (ETS) which could drive current into the tissue while simultaneously measuring
the mechanical response of the tissue. Initial validation was done by mounting the
probe in a DynaStat chamber and performing tests on excised bovine cartilage disks.
Initial results of current generated stress from these experiment corresponded with
+
+
Electroosmotic
Flow
SElectrophoretic
Force
Figure 2-3: Current Generated Stress (CGS). The negative fixed charges on the ECM
move toward the positive electrode while the ions in the fluid move toward the negative
electrode. By driving a sinusoidal current, the ensuing motions of the fluid and solid
phases create a mechanical stress within the cartilage that can be measured at the
surface.
Load cell mount
Upper
electrode contact
Porous platen
Recirculation
ports
Cartilage sample
Ag/AgCI electrode
SLower electrode contact
Actuator mount
Figure 2-4: Cartilage is uniaxially confined between two silver electrodes to produce
current generated stress.
I
IL-X/2
SENSOR
0-0.2 -0.4
CARTILGE,,
-
-0.6 -
-0.8 -
ILAG ----------------- -IAR
-1 I
I
I
I
-0
-2.5
-2
-1.5
-1
-0.5
I
0
0.5
1
I.5
1.5
2
2
2.5
2.5
<- Surface -+
Figure 2-5: Results of a theoretical model showing the flow of the current density
coming from two electrodes on the same side of the testing surface.
those predicted by the poroelastic theory. A schematic of the experiment setup is
shown in Figure 2-7.
+++
+--
+
++-
+-lil
+
LONG
WAVELENGTH
+
+
-
(B)
+-
"VERY LONG"
WAVELENGTH
(C)
Figure 2-6: A variable wavelength in-vitro probe. Different polarity configuration
give different wavelengths and penetrate at various depths of the tissue. [Courtesy of
S Berkenblit]
Collet - to DynaStat load cell
Ltion electrode inputs
Stress Sensor outputs
Bath recirculation
ports
re disc (or rubber for calibration)
-
Collet - to DynaStat actuator
Figure 2-7: An experimental setup to test the handheld probe on a disc of cartilage.
[Courtesy of D Bombard]
The present project focused on testing an in-vivo probe on an intact human
joint. The issues of in-vivo testing were addressed along with the methods for evaluating the measurements. Also, correlations of CGS measurements with mechanical
parameters and biochemical results were performed. In addition, the design and construction of a new handheld in-vivo probe was completed. The new version incorporated both the in-vivo properties of the previous handheld probe and the advantages
of using multiple electrodes. Also, a reduction in the probe diameter, to the size of
an arthroscopic instrument, was made in the new version. Finally, the setup for the
probe and its hardware components was assembled for future experimentations.
Chapter 3
Design and Manufacturing
The design of the probe took into consideration a set of requirements that would
likely be encountered in a arthroscopic surgical setting. The first requirement of the
probe is to be sufficiently small enough to enter into a joint cavity like the knee.
Secondly, parts of the probe must be easily replaced or repaired. And finally, the
measurements must be translatable into parameters that the end user (the surgeon)
could understand and incorporate into a diagnosis.
The current version of the handheld probe (v5.0) improved on the original
version (v4.0) in several ways. One significant improvement was the decrease in the
outer body diameter by 55% to 4.5 mm (compared to 1.0 cm in the v4.0). Also the
v5.0 utilized variable wavelengths to probe beneath the cartilage surface.
The design of the v5.0 probe was proposed by Steven Treppo and manufactured
with the aid of David Breslau (Center for Space Research, Cambridge, MA). In this
chapter the design and development of the probe to a working instrument is presented.
Additional details of the design specifications for the v4.0 and v5.0 probe can be found
in Appendix A and B, respectively. The original design of the v4.0 probe is presented
in David L Bombard's Masters thesis at MIT(1995).
Probe Construction
3.1
The body of the probe is comprised of three parts (Figure 3-1)
(1) Inner core
(2) Insulating sheath
(3) Outer stainless steel body
A
B
Figure 3-1: Three parts of the version 5.0 probe. A - Inner core, B - Insulating sheath,
C - Outer stainless steel body.
3.1.1
Inner Core
The inner core houses the wires that connect to the Electrode Transducer System (ETS). The ETS is the component which makes contact with the cartilage surface.
The ETS is responsible for driving current into the tissue and sensing the current generated stress response. The components of the ETS will be discussed in full detail in
the next section.
The cylindrical steel inner core is hollow (Figure 3-2A), allowing for wires to
pass through it. On top of the steel core is a cylindrical torlon body (Figure 3-2B).
Four copper tabs come out of the torlon's side and into their respective recesses.
Fitted at the end of the torlon body is a head comprised of a steel shell with an
inner area filled with a non-conducting two part epoxy. Within the epoxy, four 0.03
inch diameter brass rods are potted into positions at 90 degree intervals along the
periphery of the head. The rods make contact with the piezo electrodes of the ETS
(see section 3.2.4) and send the signals from the piezo electrode to wires in the core.
The steel shell acts as a conductive ground plane that sends extraneous signals to a
metal wire connected to ground. A crucifix pattern is machined into the hardened
epoxy to enable alignment of the ETS during the mounting phase.
There are two sets of wires that pass through the inner core. The first set of
wires are four individually insulated wires that carry the current that is driven into
the tissue. Another set of four insulated wires are bundled together by a insulated
steel mesh wrapping. These four wires send the response signal from the current
generated stress of the tissue. The steel mesh acts as the connection to the ground
plane of the steel shell.
3.1.2
Insulating Sheath
The insulating sheath is a thin cylindrical shell made of plastic torlon. The
sheath is fitted over the ETS that is placed over the end of the inner core (Figure 33). The end of the sheath is open, exposing the surface of the ETS. The sheath is long
enough to cover the copper tabs on the side of the torlon body to prevent its contact
with the stainless steel outer body. In addition the edge of the sheath is angled to
press fit the ETS over the rim of the inner core. The contacts between the silver
electrode arms of the ETS and the copper tabs of the inner core are also stabilized
by the sheath.
Brass Rods (4)
Hardened Epoxy-
H
-
.
Crucifix recess
B
A
Figure 3-2: Diagram of the inner core. A - Hollow stainless steel tube, B - Torlon
body.
Insulating Sheath
ETS
Torlon Body
Figure 3-3: Diagram of the sheath placed over the ETS and head of the inner core.
3.1.3
Outer Stainless Steel Body
The outer body is a cylindrical stainless steel tube that acts as a stiff cover to
protect the inner components of the probe. One end of the outer body is open to
expose the surface of the ETS but angled to catch the edge on the end of the probe.
The other end of the outer body is flared outward. The outer body is slid over the
sheath/inner core. A nut is then slipped over the outer body, making contact with
the threads on the inner core while pulling down upon the flared end of the outer
body. As the nut is screwed, the outer body is tightened over the sheath/inner core
(Figure 3-4).
3.2
Fabrication of ETS
The Electrode Transducer System or ETS is the element of the probe that
delivers the current into the tissue and subsequently senses the mechanical stresses
I
Outer Body
Placed
Over
Figure 3-4: Diagram of the outer body covering the probe's interior. A nut screws
the probe parts tightly together.
from the tissue. The ETS is a 100 /pm thick wafer with silver electrodes on one
side and a piezoelectric kynar electrodes on the opposite side. The silver electrode
helps deliver the current into the tissue surface. Because the ETS is thin, the current
generated stress from the tissue is felt by the kynar electrode.
The piezoelectric
property of the kynar converts mechanical stresses into an electrical voltage. This
voltage is proportional to the amount of stress experienced by the kynar film. This
output from the kynar is the parameter by which we measure the current generated
stress of the tissue.
The fabrication process of an ETS is composed of 5 phases.
(1) Assembly
(2) Photofabrication
(3) Etching
(4) Cutting and Mounting
(5) Fabrication of Silver/Silver Chloride Electrodes
3.2.1
Assembly
The assembly begins by cutting the material to their appropriate sizes. The
materials to be bonded together are an 18 mm x 18 mm piece of 25.4 /m thick silver
foil (Johnson Matthey, Ward Hill MA), an 11 mm x 11 mm piece of 25.4 upm thick
Mylar polyester film metallized only on one side with aluminum (MADICO, Woburn
MA), and a 4.5 mm diameter circular punch of a 52 am thick PVDF (Polyvinylidenefluoride) piezo film called Kynar (AMP Inc., Norristown PA) [11]. After cutting,
the Kynar and silver pieces are washed in a detergent solution (PEX) and rinsed
in deionized water to remove any residues. The silver is then dipped in 15% nitric
acid for 30 seconds and rinsed in deionized water. A two-part urethane epoxy Tycel
7000/7200 (Lord Corp, Erie PA) is mixed at a 50:1 by volume ratio and diluted with
methylethylketone at a 1:1 by volume ratio. The epoxy mixture is then applied to
the unmetallized side of the Mylar. After letting the epoxy evaporate for 15 minutes,
the Mylar is bonded to the silver. Next silver epoxy TRADUCT2902 (TRA-CON
Inc., Medford MA) is applied to the unstriated side of the piezo film and then bonded
to the metallized side of the Mylar. The assembled ETS is then pressed together
between towels in a vise for an hour. The resulting product is stored overnight to
cure for the next phase (Figure 3-5).
Silver Epoxy between Kynar and Mylar
Kynar Film
Tycel Epoxy between Silver and Mylar
,,F
I
- Metallized Mylar
If
-
'
Silver Foil
Striated Side of Kynar Up
18 mm
Metallized Sideof Mylar
18 mm
Figure 3-5: Dimensions of each layer of ETS materials.
3.2.2
Photofabrication
The purpose of photofabrication is to place the patterns of the electrodes on
both sides of the ETS. First the ETS is baked at 800 C for 10 minutes to remove any
moisture. Then in a darkroom, KPR Photoresist (KTI Chemicals, Sunnyvale CA)
is applied on both sides of the ETS and left to dry hanging for 30 minutes. The
ETS is baked again at 800 C for 10 minutes. The ETS is then placed between two
registered masks that contain the patterns of the electrodes (Figure 3-6). The masks
with the ETS are exposed to UV light for 15 minutes. The translucent patterns on
the masks enable the exposed photoresist to crosslink. The various versions of the
electrode patterns are presented in Appendix B. Afterwards the ETS is dipped in a
xylene based KPR Developer (KTI Chemicals, Sunnyvale CA) bath for 30 seconds
and then into another bath with fresh developer for 30 seconds. This step washes off
the uncrosslinked photoresist surrounding the electrode patterns. The ETS is then
rinsed with warm running water and then with deionized water.
NW
l
T
Figure 3-6: Patterns for the electrodes. Left - Piezo electrodes, Right - Silver electrodes. [Courtesy of S Treppo]
3.2.3
Etching
Etching is performed on the ETS to dissolve away areas of the silver and metallization on the piezo film not protected by the crosslinked photoresist. The ETS is
sealed in a holder with a well that exposes the silver side of the ETS. 55% w/v ferric
nitrate etchant at 45 0 C is placed in the well. The holder is stirred to help etch the
silver material. Used etchant is replaced by fresh etchant every 2 minutes. After all
the silver around the electrode pattern is removed, the ETS is rinsed with deionized
water. To etch the piezo side of the ETS, the etching process is confined within
the 4.5 mm diameter circle of piezo film with etching taking only 10 seconds. After
etching both sides, the remaining crosslinked photoresist is removed by wiping it off
with a cotton swab dipped in KPR developer solution. After rinsing with deionized
water, the result is an ETS with silver electrodes on one side and piezo electrodes on
the other.
3.2.4
Cutting and Mounting
The next step in ETS fabrication involves cutting the ETS into a pattern that
enables it to be fit onto the head of the probe. During the photofabrication step, the
outlines of the border were also marked onto the ETS. Cutting is performed along
the borders with a sharp scalpel (Figure 3-7). After cutting, a 0.33 mm thick crucifix
shaped plastic backing plate is attached to the piezo side of the ETS by a 2-part epoxy
and dried overnight (Figure 3-8). The backing plate helps with the alignment of the
ETS onto the probe and ensures it to be flat. The head of the probe has a machined
recess in the shape of the crucifix so that the ETS can fit with the proper orientation
to line up the electrical contacts. The brass contacts at the head of the probe receive
signals from the piezo electrodes while the copper tabs on the side of the torlon
body connect with the arms of the silver electrodes. The current is driven through
wires leading up to the copper tabs and onto the silver electrodes while the current
generated stress is transferred to the piezo electrodes and transmitted through the
brass contacts to the output wires. Once contacts have been made, the torlon sheath
is fitted over the head of the probe, making sure the ETS is lying flat on the surface
of the head. Before the torlon sheath is completely fitted over the head, electrical
connections are tested to the silver electrodes. Once all contacts are established, a thin
layer of waterproof RTV108 silicon rubber adhesive (GE, Waterford NY) is placed
on the inside edge of the torlon sheath. Finally the outer stainless steel cylindrical
body is slipped over the probe. Adhesive is also placed on the inside edge of the outer
body before the probe finally assembled. Excess adhesive is wiped off the edges. The
sealed probe is allowed to dry for at least 24 hours.
Metallized Mylar Areas
Silver Electrodes
Piezo Electrodes
0 25 inch
Figure 3-7: After etching and cutting the ETS. Left - Kynar electrodes, Right - Silver
electrodes.
3.2.5
Fabrication of Silver/Silver Chloride Electrodes
Chloridation of the silver electrodes facilitates the transfer of current onto the
surface being tested by lowering the interfacial impedance of the electrode/electrolyte
interface. Chloridation is performed by placing the head of the probe into a 0.1 M
NaCl solution. A closed loop electrolytic cell is produced with the silver electrode as
the anode and a platinum strip placed in the bath as the cathode. A current is driven
through the leads to deposit chloride ions onto the silver electrode. The total amount
of chloride deposition was set at 1000 mAsec/cm 2 . So given an electrode area of 1.59
mm 2 the time of chloridation and amplitude of current can be varied according to
the deposition amount. The process is repeated with each of the four silver electrodes
until they are all chloridated (Figure 3-8). A picture of the fully developed probe is
shown in Figure 3-9.
H
Backing Plate
Chlonded Electrodes
Figure 3-8: Left - Crucifix backing plate attached to kynar side of ETS, Right - Silver
electrodes after chloridation.
Figure 3-9: Schematic of proposed measurements in an joint cavity, Right - Picture
of an assembled v5.0 probe.
43
Chapter 4
Experimental Method
Sixty human patella joints were donated by the Rush-Presbyterian Hospital
(Chicago, IL) for this study. The patellas varied in gender, age, race, and Collins
grade. Each patella was sent in individual containers and frozen at -20 0 C. The version 4.0 (v4.0) probe was used to test eight patellae at two sites on the cartilage
surface. The probed sites were then excised for electromechanical testing by confined
compression. These measured parameters were compared with probe measurements
to determine if any correlations existed between the two types of measurements. In
addition, biochemical composition measurements were performed on the tested tissue
to relate the probe and electromechanical measurements with biochemical composition. The goals of the experiment were to demonstrate the ability to use the probe
on human tissue, to identify differences between tissue grades, and to detect changes
between tissue that appears to have the same visual grade.
Also included in this chapter is a synopsis of the validation tests performed on
the version 5.0 (v5.0) probe. Like the earlier stages of the v4.0 development, the
v5.0 was extensively tested in a controlled environment using excised slices of bovine
tissue. Validation of the v5.0 probe will enable its use on the remaining human patella
joints.
4.1
Calibration
Calibration was performed in order to correlate the signal output of the piezo
electrode to a known mechanical stress. This was done to convert the output during
current generated stress experiments to a mechanical stress. Calibration also gave
reassurance that the connections between the piezo electrodes and the brass contacts
were reliable as well as to ensure proper sealing. An incorrectly sealed probe would
enable seepage of the buffer fluid into the probe and risk short-circuiting the system.
4.1.1
Hardware setup
Outputs from the probe through two coaxial cables (labelled left and right
channels) were attached to a two channel electrometer. The electrometer had an
impedance control box that controlled the drift of the piezo electrodes. Output from
the electrometer went through a low-pass filter (Model 1022F, Rockland Systems,
West Nyack NY) set at a cutoff frequency of 15.7 Hz. The output from the filter was
sent to both a chart recorder (Brush 2200, Gould Electronics, Cleveland OH) and an
analog to digital converter (ADC) box to a computer running the program Dynssp
(Eliot Frank, Cambridge MA). The computer program also sent signals, through a
digital to analog converter (DAC), to control a mechanical-servo material testing
device (DynaStat, IMASS, Hingham MA). The Dynastat is able to measure the load
in materials under controlled displacement.
4.1.2
Procedure
Calibration began by attaching the probe in the DynaStat. The head of the
probe was lowered into a chamber and onto a rubber disk, and a static offset stress
of 50 kPa was imposed by the DynaStat. Phosphate Buffered Solution (PBS) was
added to the chamber. There was a waiting period of fifteen minutes to let the system
thermally equilibrate. The computer program Dynssp was programmed to direct the
DynaStat in applying a dynamic stress amplitude of 10 kPa in a frequency range of
1.0 to 0.025 Hz. The procedure was repeated again at an amplitude of 2.5 kPa. The
resulting outputs signals were saved on the computer.
Current Generated Stress Experiment
4.2
Hardware Setup
4.2.1
Outputs from the probe were attached to the two channel electrometer. From the
electrometer the signal was passed through the Rockland filter. Both outputs from
the filter were fed into a differential amplifier (Model 11-4113-01, Gould Electronics, Cleveland OH). The differential signal was displayed on the chart recorder. The
differential output from the chart recorder was fed through the ADC box to the computer. Dynssp was used as the data aquisition program and controller of the current
generation. The computer sent its current controlling signal, through a DAC box, to
a current source (Kepco, Flushing NY). The Kepco was attached to a frequency synthesizer (Rockland Systems, West Nyack NY) and monitored by an ammeter (Keithley Instruments, Cleveland OH). The frequency generator sent the current to the
wires leading to the silver electrodes of the probe. The frequency generator also sent
impedance measurements to the Gould chart recorder.
Testing for Parasitic Signals
4.2.2
"Parasitic" testing was done to measure any artefactual response that may have
been induced by application of the current but not related to the mechanical stresses
within the cartilage. Testing for the parasitic response was done by suspending the
probe in buffer and applying a current into the solution. Because the solution does not
respond with a mechanical stress, the ETS should not produce an output piezo signal.
However it had been observed in past experiments that there were some parasitic
signals either created by incomplete shielding or perhaps by some other unknown
coupling phenomenon. The need to record any parasitic response was important to
correct for the actual responses when probing cartilage tissue.
4.2.3
Tissue Experiments with the Probe
Patellae were thawed at room temperature for one hour in Phosphate Buffered
EDTA (PBE) containing protease inhibitors (1.0 mM PMSF, 1.0 mM E64, 1.0 uM
pepstatin, and 1.0 uM leupeptin). Extraneous tissue surrounding the cartilage was
carefully removed with a scalpel, taking care not to injure the cartilage surface. PBE
w/inhibitors was sprayed onto the surface to keep the cartilage moist and safe from
proteolytic enzymes. The patella was then mounted in a chamber filled with the same
buffer (Figure 4-1).
Probe measurements were done at two locations on the cartilage surface. The
head of the probe was placed on a flat area of the curved cartilage surface to obtain the
most reliable measurements. The other end of the probe was placed in a cylindrical
shaft which contained a calibrated spring. The probe compressed the spring a set
distance ensuring that when the shaft was clamped to a fixture, the spring would
produce a static stress of 50 kPa onto the probe and subsequently on the tissue
(Figure 4-1).
The computer program Dynssp, which controlled the driving current, was set to
a current density of J = 0.5 mA/cm2 . The current was programmed to be applied as
a sinusoid at frequencies ranging from 1.0 to 0.025 Hz. The procedure was repeated
again at a current density of J = 1.0 mA/cm 2 . The two piezo electrodes sensed the
mechanical stresses from the tissue and each produced an output voltage. The signals
were then processed and recorded by Dynssp.
Confined Compression
4.3
4.3.1
Hardware Setup
A confined compression chamber which holds tissue samples was mounted in the
DynaStat. The bottom of the chamber contained a silver electrode. Another electrode
was suspended in the bath solution. The electrodes measured the streaming potential
Figure 4-1: Setup for an intact joint experiment. Patella is placed in a holder and
submerged in testing solution. Probe is inserted into a probe sheath clamped to a
ring stand. The end of the probe is placed on a flat surface of the joint. A calibrated
spring in the shaft provides the required static offset stress.
48
that occurred during dynamic tissue compression. The signal from the electrodes was
fed to a chart recorder and a computer running Dynssp. Dynssp also sent signals
via a DAC to the DynaStat in order to control the displacement of the metal platen
that was applied to the tissue. The load and displacement signals were sent from the
DynaStat to the chart recorder and computer.
4.3.2
Procedure
After testing with the v4.0 in-vivo probe, the identical sites in contact with
the probe were drilled and a 9.5 mm diameter core of cartilage/bone was extracted
from the joint. Two cores were taken, one from each site probed with the articular
surface intact. A 1.0 mm thick slice of the cartilage surface was microtomed from
the core and placed in a 0.15 M NaCl/Trizma buffer. From each slice, four 3.0 mm
diameter disks were cut out with a biopsy punch (Figure 4-2). The thickness of one
disk was measured and placed on the silver electrode in the confined compression
chamber (mounted in the DynaStat) with the articular surface facing away from
the ground electrode. A porous platen was placed on top of the disk and a metal
platen was lowered onto the porous platen. Buffer was poured into the chamber
and the other silver electrode was submerged into the bath. The cartilage disc was
compressed to various strain levels (10%, 15%, 20%, 25%) and allowed to stress
relax for 400 seconds at each level. At a 15% strain, a 0.5% dynamic compressive
strain was applied and the resulting load and streaming potential were measured at
frequencies between 1.0 to 0.01 Hz. The equilibrium modulus, hydraulic permeability,
and the electrokinetic coupling coefficient of the tissue were calculated from these
measurements using previous established methods [18, 19].
4.4
Biochemistry
The remaining 3.0 mm diameter disks were kept for biochemical measurements.
The samples were first weighed wet and then re-equilibrated in a hypotonic solution
of 0.01M NaCl for two hours to induce swelling. The samples were then weighed again
Patella Joint
T
O
U
0
Mechanically Tested
via
Confined Compression
Un tested
#1
Cut Out
#2
9 mm Diameter Core
Biochemistry Testing
O
- Swelling
- GAG
*
Microtome 1 mm
Thick Slice of
- DNA
#3 QQ
- Hydroxyproline
4
Punch Out Four 3 mm
0
Articulating Surface
Diameter Disks
9 mm Diameter Plug
C
Figure 4-2: Test sites were drilled and a 9.5 mm diameter core was extracted from
the joint. A 1.0 mm thick slice of the cartilage surface was microtomed from the core.
From each slice, four 3.0 mm discs were punched out. One disc was tested in confined
compression while the others were tested for biochemistry.
(called a swell weight). Samples were subsequently placed in individual cyrovial tubes
to be lyophilized 48 hours to remove all water from the tissue. The samples were then
weighed dry. The water content of the tissue was found by calculating the difference
in wet and dry weights. Next, papain solution was added to digest the samples down
to its individual biochemical components. The digested samples were then analyzed
for GAG content (via a DMB dye binding assay using a spectrophorometer) [14],
DNA content (using Hoechst dye 33258 with a spectroflourimeter) [28], and collagen
content by measuring the amount of hydroxyproline present [53].
Version 5.0 Validation
4.5
4.5.1
Calibration
The usual calibration setup was repeated with the only difference being that all
four channels were individually calibrated. The output wire from the probe went to
a split box which separated each channel to an individual coaxial cable output. With
the two input electrometer, two piezo sensors were calibrated at a time. In addition,
the filter was set to give the input signal an external gain of 20dB (or 10 times).
4.5.2
Procedure
Calf knee joints were delivered within 24 hours of slaughter (Research 87, Boston
MA). The distal femur knee joint was dissected out and a 9.5 mm core was removed
from the femoropatellar groove. A 1.0 mm slice of articulating cartilage was microtomed off the core and placed in a chamber with the articulating side up. The
chamber and probe were mounted in the DynaStat and the probe was lowered onto
the tissue. IX PBS was added to the chamber and an offset stress of 50 kPa was
placed on the tissue. The setup for the current generated stress experiment was the
same as the v4.0, except the current from the current generator goes through a switch
box that can vary the polarity pattern of the silver electrodes. The polarity of the four
silver chloride electrodes were varied to give the different spatial wavelengths applied
to the tissue (either short, long, or extra long). In the validation, only short and extra
long wavelengths were used. For the short wavelength configuration, the polarity for
the channels was 1 + 2 - 3 + 4 - (Figure 4-3). The area of a silver electrode was
1.59 mm 2 , so for a current density of J = 0.5 mA/cm 2, the amplitude of the driving
current was 15.9 /A. The current was split between the two positive electrodes. For
J = 1.0 mA/cm 2 , the amplitude was 31.8 pA. Because the electrometer could only
measure two electrodes at a time, the test was repeated twice. The first run tested the
differential output between channels 1 and 2, and the second run between channels
3 and 4. For the extra long wavelength configuration, the only channels used were 1
+ and 4 - (Figure 4-3). Therefore for J = 0.5 mA/cm 2 , the driving amplitude was
8 AA, and 15.9 ALA for J = 1.0 mA/cm 2 . The extra long test required one run with
outputs coming from channels 1 and 4.
Parasitic tests were also done using the previous technique. Parasitic runs were
performed using the short wavelength configuration at J = 0.5 and 1.0 mA/cm 2
Again only two channels could be measured at a time
2/2
+
+
(A)
Cartilage
I/2
++OO
0 0
(B)
Cartilage
Figure 4-3: Polarity configurations for A - short and B - extra long wavelengths. 0
in electrodes 2 and 3 for (B) denote no current output. The longer wavelength in (B)
penetrates deeper into the cartilage tissue.
Chapter 5
Results
Results from the three types of patella experiments (probe, confined compression, and biochemical) are presented to highlight the relationships between these
measures. In addition, both the mechanical and biochemical results are compared
with previous studies on other joints. Next, data from the patella and femoral patellar groove joint surfaces, which are in contact with each other, are compared. Finally,
results from an experiment utilizing the v5.0 probe are presented regarding validation
of the probe as a working device for future experiments.
5.1
Patella Experiments
Before experimentation, the right and left piezo channels of the v4.0 probe were
calibrated with known dynamic stresses. In general, the outputs from each channel
were similar in magnitude. A typical working probe gave a calibration response as
shown in Figure 5-1. The piezo output increased with an increase in dynamic amplitude and also with a decrease in frequency. Typically the phase angle of the output
signal was approximately 180.0 degrees, with the angle decreasing with increased
frequency.
The differential output in mV was measured at two sites on eight patellae joints
for a total of sixteen measurements. These measurements were then converted to
Right Piezo Channel
Left Piezo Channel
500
500
O 0.025 Hz
400
400
X 0.05 Hz
O 0.1 Hz
300
300 -*0.25 Hz
A 0.5 Hz
200
200
+ 1.0 Hz
-
100
100
- /0
m
0
-
5
2.5
r
7.5
10
-
0
12.5
5
7.5
10
12.5
5
7.5
10
12.5
200
200
1L
150
~------.
150
100
100
50
50
0
2.5
m
0
2.5
5
7.5
STRESS, kPa
10
12.5
0
2.5
STRESS, kPa
Figure 5-1: Calibration outputs from the left and right channels of a v4.0 probe.
current generated stress (CGS) results by using the calibration for that particular
experiment.
For some tests the parasitic response was significant before and after the testing
of a site. In order to correct the CGS measurement, an average of the parasitic
signal before and after the site measurement was calculated. This average parasitic
response was then subtracted from the output voltage at that site. Statistical outliers
or corrected measurements whose values were negative were removed from the data
pool. Data from patellae having Collins grade 0 (i.e., normal) are reported below
(these data correspond to eight tested sites).
The averaged CGS along with the
corrected CGS are shown in Figure 5-2. The graph shows that CGS decreased with
increasing frequency in both cases. This result correlates with theoretical predictions.
* Total output signal
I CGS (total signal
minus no-tissue
baseline)
0.4
0.3 -
0.2 -
0.1 _MEAN +/- SE (n=8)
0.01
I
I
0.1
Frequency (Hz)
1
Figure 5-2: Current generated stress versus frequency, N = 8.
The results of confined compression and biochemical composition measurements
are shown in Table 5-1 for these same 8 patellae.
Mechanical Properties (N=8)
Units
Mean
Std Dev
Dynamic Stiffness @1.0 Hz
MPa
3.23
1.43
Dynamic Stiffness @0.1 Hz
MPa
2.51
1.10
Dynamic Stiffness @0.01 Hz
MPa
1.68
0.65
Streaming Potential @1.0 Hz
mV/%
0.09
0.07
Streaming Potential 00.1 Hz
mV/%
0.07
0.05
Streaming Potential @0.01 Hz
mV/%
0.04
0.03
MPa
0.41
0.22
Stiffness Coefficient Ks
MPa/mm
3.04
1.24
Electromechanical Coupling Coefficient Ke
mV/MPa
1.16
0.67
(p m/s)/(MPa/mm)
6.64
3.57
mm
1.37
0.28
Water content (% of wet weight)
%
78.42
2.96
GAG Content (% of dry weight)
%
8.32
4.11
GAG Content (% of wet weight)
%
1.78
0.92
DNA Content (% of dry weight)
%
0.11
0.04
DNA Content (% of wet weight)
%
0.02
0.01
Hydroxyproline Content (% of dry weight)
%
9.21
3.76
Collagen Content (% of dry weight)
%
65.73
26.85
Collagen Content (% of wet weight)
%
14.35
6.75
GAG/DNA ratio
ug/ug
139.91
92.24
GAG/Collagen ratio
ug/ug
0.17
0.15
cells/g x10 6
29.95
9.67
Equilibrium Modulus HA
Permeability K
Thickness
Biochemical Composition (N=8)
# Chondrocytes per gram of tissue
5.1.1
Correlations
The results from the three types of experiments were compared to determine
if any parameter correlations exist. First a correlation was performed between CGS
measurements and electromechanical/biochemical composition results (Figure 5-3).
0.1 R= 0.6408 (n = 6)
A
B
R= -0.8326 (n = 6)
0.05
0.1
o
0.05N
N
"1
n,
0
0
0.2
0.4 0.6
HA (MPa)
0.8
0.1 R= -0.4690
(n = 6)
1
0
25
50
75
age (years)
D
R= 0.8347 (n = 7)
C
0.5 0
0
*S
0.05
*
0.25$
N
-U
n
S
11J
0
2.5
5
7.5
10
k (gm/s)/(MPa/mm)
12.5
75
I
80
H2 0 Content (%)
85
Figure 5-3: Correlations of the current generated stress with A - equilibrium modulus,
B - age, C - permeability, and D - water content.
From the data, there was a high correlation between CGS (at 1.0 Hz) and age,
and also between CGS (at 0.025 Hz) and water content (Figure 5-3B,D). Correlations
were done using a Pearson's correlation [48]. CGS decreased with increased age (R
= -0.83) and increased with increased water content (R = 0.83) (Figure 5-3B,D) . A
lower correlation was seen when comparing the CGS (at 1.0 Hz) to the equilibrium
modulus (HA) (R = 0.64) and permeability (k) (R = -0.47), where HA and k were
obtained from the confined compression tests. This suggested that the HA goes up
while k goes down with increased CGS (Figure 5-3A,C).
Correlations were also found within the electromechanical results. For example,
the electromechanical coupling coefficient (ke) increased as the streaming potential
(R = 0.98) and dynamic stiffness (R = 0.95) each increased (Figure 5-4). This trend
occurred at all frequencies measured for both the streaming potential and dynamic
stiffness.
2.5 R= 0.9788 (n = 8)
*
2 1.5.r 0.5 -
.
S0
*
R= 0.9463 (n = 8)
*
*
I
A
0.2
0.15
0.1
0.05
0
Stream Pot @ 0.1Hz (mV/%)
.
0
2.5
*
B
-1.5
3
- 0.5
=
5
4
3
2
1
Dyn Stiff @ 0.1Hz (MPa)
Figure 5-4: Correlations of the electromechanical coupling coefficient with A - streaming potential and B - dynamic stiffness.
Regarding biochemical composition, as age increased, water content (R = -0.58)
and GAG per wet weight (R = -0.58) decreased (Figure 5-5B,C). In addition, the
hydroxyproline per wet weight ratio decreased with increasing HA (R = -0.56) (Figure 5-5D), and HA was found to decrease with age although the correlation coefficient
was lower at -0.30 in these grade 0 tissues (Figure 5-5A).
5.1.2
Joint Comparisons
In previous experiments, electromechanical and biochemical composition measurements were performed on human knee and ankle joints using the same protocols
as described in the last chapter. A comparison of these results with those obtained
from the patella will be useful in determining what similarities or differences exist
between the various cartilages from different joint surfaces [51].
1.2
85
R= -0.5828 (n = 8)
R= -0.3045 (n = 8)
I
o
0.
80 o0
* 0
20.6
*' S
0*
I
-
0
I
I
o
I
75
75 .
A0
0(
0
25
50
age (years)
75
0
50
25
age (years)
R= -0.5629 (n = 8)
R= -0.5848 (n = 8)
70
75
5
I
4
"
O
3
0
I
"
0 o0
I
25
50
age (years)
2
75
0
0.5
HA (kPa)
Figure 5-5: Correlations of age with A - equilibrium modulus, B - water content,
and C - GAG per tissue wet weight, also D - correlation of equilibrium modulus to
hydroxyproline content.
The first set of results compare the electromechanical properties of patellar (PT)
cartilage to talar (TA), distal femur (F), and tibial plateau (TP) cartilage. Several
differences were observed using a student t-test analysis assuming equal variances
with p < 0.05 being significant and p < 0.01 being highly significant. First, the
pooled HA, dynamic stiffness, and k of the patellar cartilage was different than talar
cartilage. Like other knee cartilage surfaces (F and TP), the HA (p < 0.05) and
dynamic stiffness (p < 0.01) of patellar cartilage was lower than talar cartilage while
the permeability in patellar cartilage was higher (p <0.01) (Figure 5-6A,B,C). Of note,
one patellar sample did have an unusually high HA (over two standard deviations
from the mean) that significantly influenced the average HA result. Interestingly,
except for a difference in dynamic stiffness (p < 0.01) from the distal femur, the
electromechanical properties of patellar cartilage were similar to the properties of
cartilage from the distal femur and tibial plateau.
With regards to the biochemical composition, the patellar cartilage also showed
some differences when compared with ankle and knee cartilages. The water content
of the patellar cartilage was higher than both the talar (p < 0.01) and distal femur
(p < 0.05) cartilage. Another significant variation was in the swelling ratio, defined
as the swell weight divided by the wet weight. The patellar cartilage swelled more
than any of the other cartilage surfaces (p < 0.01 against TA and F; p < 0.05 against
TP). On the other hand, there were no differences in the GAG to DNA ratio or the
hydroxyproline to wet weight ratio amongst the various cartilage. (Figure 5-7).
5.1.3
Comparison of Articulating Cartilage Surfaces
Comparing the properties of cartilage tissue in contact between two articulating
joints was of interest with respect to how their properties matched. The electromechanical results and the biochemical composition of cartilage from the patellar surface
were compared with those from the distal femur surface. Comparisons were first made
between the entire pooled data sets of the patella and distal femur joints (denoted
as FP and PT in Figures 5-8,9). Then the patellar and femoropatellar data which
1.2
Equil. Modulus (MPa)
10
18
TA
F
TP
PT
k (gm/s)/(MPa/mm)
0
0.1
PT
F
TP
TA
Stream. Pot. 0.1Hz (mv/%)
D
T
0.05
9
0
B
5
0.6
0
Dyn. Stiff. 0.1Hz (MPa)
TA
F
TP
PT
* p<0.01 , p<0.05,TA vs F TP PT
* p<0.01,] p<0.05, F vs TP ,PT
0
TA
F
TP
PT
0 p<0.01,O p<0.05, TP vs PT
Figure 5-6: Comparing electromechanical results of cartilage from various joint surfaces. Cartilage came from TA - talar, F - distal femur, TP - tibial plateau, and PT
- patellar joint surfaces.
H20 Content (%)
85
GAG / DNA (gg/gg)
150
B
80
75
-
75
70
3
TA
C
- lf
1.5
0
F
TP
PT
Hypro / wet wt. (%)
TA
F
TP
0
1.08
1.03
PT
0.98
* p<0.01 * p<0.05,TA vs F TP PT
M p<0.01 , p<0.05, F vs TP,PT'
PT
TP
F
TA
Swelling Ratio (0.15M/0.01M)
D
[-1
TA
F
TP
PT
* p<0.01,0 p<0.05, TP vs PT
Figure 5-7: Comparing biochemical properties of cartilage from various joint surfaces.
Cartilage came from TA - talar, F - distal femur, TP - tibial plateau, and PT - patellar
joint surfaces.
both came from the same human subjects were extracted from the pooled data and
compared (denoted as FP-C and PT-C in Figures 5-8,9). In this extracted data, two
sets of distal femurs and patellae were compared (N = 4).
Results from comparing surfaces in contact showed no difference in electromechanical and biochemical parameters (Figures 5-8,9). The only significant difference
was in the swelling ratio. When comparing the entire set of patellae and femoral
samples, the swelling ratio of patellae was much higher between the two sites (p <
0.05) (Figure 5-9). More samples will be needed to establish more robust data.
1.2
Equil. Modulus (MPa)
5
0.6
0
10
FP
18
PT
FP-C PT-C
k (gm/s)/(MPa/mm)
0
0.1
Dyn. Stiff. 0.1Hz (MPa)
T
PT
FP-C PT-C
FP
Stream. Pot. 0.1 Hz (mv/%)
0.05
9
=~
_-~
A
FP
PT
FP-C
PT-C
0
FP
PT
FP-C
PT-C
Figure 5-8: Comparing electromechanical results of cartilage from surfaces on the
patella that are in contact with cartilage from surfaces on the distal femur. Comparison of data from the same human subjects are denoted by FP-C and PT-C.
H2 0 Content (%)
85
80
-
GAG / DNA (gg/gg)
150
T
75
75
-c
70
FP
3
PT
FP-C PT-C
Hypro / wet wt. (%)
0
1.08
1.03
1.5
-L
0
FP
PT
FP-C
PT-C
0.98
FP-C PT-C
PT
FP
Swelling Ratio (0.15M/0.01M)
r-
*
F
fr IIi-Li
FP
PT
FP-C
PT-C
* p<0.01 ,* p<0.05, FP vs PT
Figure 5-9: Comparing biochemical composition results of cartilage from surfaces
on the patella that are in contact with cartilage from surfaces on the distal femur.
Comparison of data from the same human subjects are denoted by FP-C and PT-C.
Version 5.0 Results
5.2
The calibration of the v5.0 probe produced output signals below lmV/kPa per
piezo channel. This is due to the smaller electrode size with respect to the v4.0.
However the output followed the expected trends that were previously seen for the
piezo response. For example, the calibration signal increased with increasing dynamic
amplitude and decreasing frequency (Figure 5-10). The majority of the v5.0 ETSs
installed have had piezo channels whose signals had slight variations amongst each
other. As is shown in Figure 5-10, the output from channel 2 was higher than those
from channels 1, 3, and 4. With some ETSs, all four channels have had approximately
equivalent signals, but it has not been achieved consistently.
5
5
00.1 Hz
A 0.25 Hz
O 0.5 Hz
'3
-*1.0
Al
E3
-
Hz
111
30
0
0
v
0
2.5
5
7.5
10
0
12.5
5-
5-
4
4
3-
3-
2
2
1
00
1
00
2.5
5
7.5
10
Dynamic Stress Amplitude, kPa
12.5
2.5
5
7.5
10
12.5
2.5
5
7.5
10
12.5
Dynamic Stress Amplitude, kPa
Dynamic Stress Amplitude, kPa
Dynamic Stress Amplitude, kPa
Figure 5-10: Piezo response for each channel at various dynamic amplitudes and at
various frequencies.
Several excised bovine disks were tested for current generated stress. While
driving current in a short wavelength configuration, the differential response from
piezo channels 1-2 and 3-4 were recorded (Figure 5-11). The differential output increased with increasing current density (J) and decreased with increasing frequencies
as is expected from theory and previously collected data. The results showed that
the differential response from channels 1-2 were about an order of magnitude lower
than the output from channels 3-4. In addition, the phase angle of channels 1-2 were
more scattered compared to channels 3-4.
Channel 3-4
Channel 1-2
>
E
1.5
1.5
0 0.1 Hz
A 0.25 Hz
00.5
01
Hz
*1.0
I-
O
0
O
0.5-
0.5 0
0
0
o 200
z
1
Hz
0.5
1
1.5
2
0
2.5
0.5
1
9
9
1.5
2
2.5
200
S150-
150-
100
100-
50
50
00
-50S-50
:
t-100
ne-100
2.5
2
1
1.5
0
0.5
2.5
1.5
2
1
0
0.5
2
CURRENT DENSITY, mA/cm 2
CURRENT DENSITY, mA/cm
Figure 5-11: Differential amplitude and phase from channels 1-2 and channels 3-4
at various applied current densities and frequencies. Driving current is in a short
wavelength configuration.
The calculated CGS, using calibration results, showed similar trends to the differential piezo output (Figure 5-12). These results show the importance of observing
the individual responses of each electrode as opposed to averaging the signals, which
was the method used in past experiments involving multiple interdigitated electrodes
[3]. In addition, the results presented were not the norm for all the other experiments
made by the v5.0 probe.
6
5
04
1
0.1 Hz
A
6
54321-
--
0
A 0.25 Hz
4
0 0.5 Hz
*1.0 Hz
W
C
() 20
O
0
2.5
2
1
1.5
0.5
0
2
mA/cm
CURRENT DENSITY,
11
1
1.5
1
0
0.5
2
2.5
CURRENT DENSITY, mA/cm 2
Figure 5-12: CGS output from A - channels 1-2 and B - channels 3-4 at various
current densities and frequencies.
Measurements were also performed while driving a current with an extra long
wavelength configuration. The CGS was sensed by piezo electrode channels 1-4. The
differential output from channels 1-4 and the calculated CGS along with the phase
were consistent with previous results with the v4.0 probe (Figure 5-13).
Calculating the normalized stress amplitude (CGS divided by the current density J) and then plotting the results versus frequency, the expected trend of a decreased normalized stress amplitude with increasing frequency emerges. This was
seen for both short and extra-long wavelengths (Figure 5-14).
Results from the test for parasitic signals also yielded significant results. For this
experiment the average differential output was below 50 uV/kPa for channels 1-2,
and 3-4. This amount of parasitic signal was less than 10% of the output from the
experimental run. In contrast, the v4.0 probe had parasitic signals upwards of 50%.
In addition, the phase angle of the parasitic signal was erratic, showing no pattern
1.5
0.1 Hz
A 0.25 Hz
1
6
5
4
3
2
A
- 0 0.5 Hz
*1.0
Hz
0.5-
1
00
2.5
1.5 2
1
0.5
0
2
mA/cm
CURRENT DENSITY,
2
2.5
1
1.5
0.5
0
CURRENT DENSITY, mA/cm 2
200
150
100
50
0
-50
-1I nn
Iv !,
oc
I
I
I
I
2.5
1.5
2
1
0
0.5
CURRENT DENSITY, mA/cm 2
Figure 5-13: A - Differential amplitude from channels 1-4 at various applied current
densities and frequencies. Driving current is in an extra long wavelength configuration. B - CGS response from an extra long wavelength configuration. C - Phase
output.
A
0.3-
B-
0.3 Z
0.2-
0.2 o
"
0.1-
0.1
0
0
I
T
0.25
0.5
0.75
FREQUENCY, Hz
1
-C
0.3
0.2
0
I
0.75
0.5
0.25
FREQUENCY, Hz
0
1
I
-I
0.1
I
0C
0
I
I
0.25
0.5
0.75
FREQUENCY, Hz
Figure 5-14: Normalized stress amplitude for a short wavelength configuration in A channels 1-2 and B - channels 3-4. C - Normalized stress amplitude for an extra long
wavelength configuration in channels 1-4.
with the change in frequency (Figure 5-15, 5-16).
Channel 3-4
Channel 1-2
1.5
1.5
0 0.1 Hz
A 0.25 Hz
_ 0.5 Hz
* 1.0 Hz
1
0.5
0.5
0
n
0
170
120
70
20
-30
-80
-130
-180
0.5
1
I
1.5
I
2
2.5
I
1
1.5
2
2.5
0.5
0
CURRENT DENSITY, mA/cm 2
0
170
120
70
20
-30
-80
-130
-180
0.5
1
El
I
1.5
2.5
2
2
2.5
1
1.5
0
0.5
2
CURRENT DENSITY, mA/cm
Figure 5-15: Parasitic response from differential outputs and phase from channels 1-2
and channels 3-4.
6
O 0.1 Hz
5 - A 0.25 Hz
A
0 0.5 Hz
4
*j 1.0 Hz
3-
2
11
-
2
2.5
1.5
1
0.5
0
CURRENT DENSITY, mA/cm 2
6
B
5
4
-
3
2
1
n
-
0
0.5
2.5
1
1.5 2
2
mA/cm
CURRENT DENSITY,
Figure 5-16: Parasitic CGS from A - channels 1-2 and B - channels 3-4.
Chapter 6
Conclusions
This last chapter will summarize some of the accomplishments made so far and
to present the next steps that are to follow in the near future.
6.1
Significance of results
By performing experiments using the v4.0 probe, we have validated previous
studies and proven how the device can be used in an in-vivo setting. The demonstration that the probe can be used on an intact joint have shown what obstacles must be
addressed during future probe experiments. Obstacles such as finding a flat surface
on the joint have motivated the developments of a newer and smaller v5.0 probe. In
addition, the first results from performing a CGS measurement in joints has yielded
preliminary results for which one can target for repeatability in the future. Results
such as a correlation of CGS with HA and water content may expose these parameters
as significant in assessing cartilage integrity.
Analysis of the electromechanical and biochemical composition results have
acted as a benchmark to validate the tissue's integrity. Through these tests, it has
been shown that patellar cartilage is much more like the cartilage it articulates with
rather than ankle tissue. This is important to consider since future in-joint procedures
with the probe may involve other knee joints.
6.2
Issues
Several issues still need to be addressed in the current probe system. For the v5.0
probe, the main concern is still the ETS and the issues related to sealing. The results
from the calibrations of numerous ETSs have yielded varying results. These problems
involve the piezo electrodes and have ranged from the electrodes sending differing
calibration responses to non-sinusoidal signals. In addition, the silver electrodes on
the ETS have occasionally experienced impedance levels much higher than theoretical
values. These problems are yet to be solved and may involve the improvement of
certain steps in the ETS manufacturing process. In the area of sealing, several ETSs
have shorted out by fluid seeping into the probe. The proposed solution for this
may be to double seal the probe with both the RTV silicone adhesive and a coat of
polyurethane sealant.
On the experimentatal side, the main issue that remains is the software and
hardware elements to acquire the outputs from the v5.0 probe. Work is now being
conducted to use the data acquisition program Labview to run the CGS and calibration experiments. The advantage of Labview is that it can acquire and process all four
outputs from the probe simultaneously. In addition, Labview can also be programmed
to provide auto-drift control on the piezo electrodes to give a more faithful CGS response during an experiment. The hardware for the experimental setup is now being
put together with the construction of a 4-channel electrometer and a DAC/ADC box
that can receive multiple inputs and outputs.
6.3
Looking Ahead
Looking ahead at the entire project, the next steps involve continued testing of
the version 5.0 probe and improving on the longevity of the ETS. In addition, the
new data acquisition hardware and Labview software will be integrated with the v5.0
probe. After the system is deemed ready through experimentation on calf cartilage,
testing of intact human joints with the v5.0 can commence. The remaining human
patellas vary in Collins grade and age, allowing investigation of their effects on CGS.
With an increased number of measurements, the initial correlations presented can be
better supported or refuted. With an increased sampling pool, models relating CGS
to electromechanical parameters or biochemical composition can be made. Progress
up to this point will then enable the justification to test the entire probe system in a
surgical setting, first with animals and then humans.
In summary the design, manufacturing, and testing of an in-vivo probe has
brought together diverse disciplines such as engineering and medicine. This project
utilizes the natural cartilage property of electrokinetics in the design of a device to
diagnose its properties. In addressing certain design criteria as a medical diagnostic
tool, a device was made and tested, validating its use. It is the hope that such a discovery will help facilitate in the diagnosis of an ailment that has and will increasingly
affect a significant proportion of the population, osteoarthritis.
Appendix A
Version 4.0 Probe
Presented here are pictures of the v4.0 probe that was designed by David L Bombard.
Several modifications were made with the v4.0 ETS. Most notably was a change in
the shape of the piezo electrodes. The current v4.0 ETS electrode patterns will be
presented here. For past versions of the ETS electrode pattern and the dimensions
of the v4.0 probe itself, the reader is highly recommended to consult with Bombard's
thesis [6].
j\
-
Screw
i
Core
ETS
Sheath
Tube
Figure A-1: Parts of the v4.0 probe being assembled. Outer body diameter of the
probe is 1.0 cm. [Courtesy of D Bombard]
+ +
I
+ +I
1 inch
Figure A-2: Current v4.0 ETS electrode pattern. Left - Piezo electrode; Right Silver electrode. Cross patterns are used to align ETS wafers during photofabrication.
[Courtesy of S Treppo]
Appendix B
Version 5.0 Probe
Presented here are the geometries of the individual parts of the v5.0 probe. In addition, the design iterations of the ETS patterns are shown and described.
0.025"
/
TOP VIEW
0.22" dia --
0.24"
0.04'
SI
0.03"
I
0 .17" dia
0.22" dia
0.4"
0.11"
SIDE VIEW
0.13" dia
1.097"
0.34"
0.23" dia! r
Figure B-1: Dimensions of Inner core of the v5.0 probe.
A
0.5"
0.005"
0.23" dia
C
3.00"
2.44"
0.06"'
0.25" dia
1.50"
0.01" -
W--
0.252" dia
--
--
Figure B-2: Dimensions of A - Bottom part of inner core, B - Insulating sheath, and
C - Outer Body of the v5.0 probe.
Figure B-3: First iteration of v5.0 ETS electrode patterns. Left - Piezo electrodes;
Right - Silver electrodes [Courtesy of S Treppo]
B.1
First Iteration of ETS Patterns
The first design of the v5.0 ETS electrodes had piezo electrodes which made
contact with the brass rods through four 0.03 inch diameter circular extensions. The
total active area of each piezo electrode was 0.63 mm 2 . This is also the same area
as each silver electrode. In general the areas between the piezo and silver electrodes
should be equal.
1
._1
l
T
Figure B-4: Second iteration of v5.0 ETS electrode patterns. Left - Piezo electrodes;
Right - Silver electrodes [Courtesy of S Treppo]
B.2
Second Iteration
A second design of the v5.0 ETS took advantage of the empty areas not utilized
by the smaller electrodes in the first design. One significant change was the expansion
of the top and bottom piezo eletrodes to cover over the brass rods. This enabled
both an increase in electrode area and the transfer of response signal to the brass
contact. The overall area for each piezo and silver electrode was increased to 1.59
mm 2 . The increased piezo area corresponded to increased signal output. The width
of the connection between the silver electrode and silver arm was increased to prevent
fracture and disconnection during the mounting phase when the arms are folded over
the head of the probe. Also patterns to assist in the cutting of the ETS were placed
on the masks.
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