The Effect of Tensile Strength Loss on Collagen
Organization in Anterior Cruciate Ligament Grafts
During Continuous Passive Knee Motion
By Team Ligament Elasticity post-Graft Surgery (LEGS)
(Ayushi Chandramani, Matthew Costales, Benjamin Garbus, Joseph Hartstein, Kelley Heffner, Rupal Jain, Kelly
Klein, Alicia McDonnell, Payal Patel, Victoria Stefanelli, Jenny Wang, Joseph Weinberg, Tina Zhang)
Table of Contents
ABSTRACT
2
INTRODUCTION
3
REVIEW OF LITERATURE
5
MATERIALS AND METHODS
A. EXPERIMENTAL SETUP
B. PRE-CONTINUOUS PASSIVE MOTION ANALYSIS
C. GRAFT PRETENSIONING PROTOCOL
D. CONTINUOUS PASSIVE MOTION TESTING
E. POST-CONTINUOUS PASSIVE MOTION ANALYSIS
F. STATISTICAL METHODS
10
10
11
13
13
14
14
APPENDICES
A. EXPERIMENTAL GROUPS
B. TIMELINE
C. BUDGET
D. ACKNOWLEDGEMENTS
15
15
16
17
18
REFERENCES
19
1
ABSTRACT
Anterior cruciate ligament (ACL) graft reconstruction relies on the effectiveness of graft
materials and surgical procedures to successfully restore knee function. Current materials and
methods fall short of providing adequate recovery and commonly result in a loss of tension
(laxity) independent of the type of graft used. Past research has shown differences in graft
performance based on graft origin: ACL donor, bone-patella-tendon-bone (BPTB), hamstring
(STG).
Further research has demonstrated a correlation between crimping patterns in the
collagen fibrils of ligaments and the ability of the graft to withstand tension. No studies thus far
have combined morphological differences between grafts (BPTB, STG, ACL) with their varying
performance in tensile tests under continuous passive knee motion (CPM). This study will
attempt to explain that relationship.
BPTB, STG and ACL grafts from five pairs of cadaveric legs will be imaged using
noninvasive optical coherence tomography (OCT). One leg from each pair will serve as a
control, and the relevant tissues will be imaged using polarized light microscopy (PLM). Each
control’s corresponding leg will then undergo CPM testing. The BPTB, STG and ACL grafts
will all be individually tested and monitored for tension loss using a materials testing system
(MTS) machine. After CPM, each graft will be reimaged with OCT as well as PLM. Results
from before and after OCT imaging, as well as morphological differences between the control
and experimental specimens, as seen through PLM, will be compared to tension performance
from CPM.
Marked differences in tension loss should stem from morphological differences between graft
types. Through this research, we hope to enable better predictions to be made regarding the
extent of joint laxity displayed by specific grafts post ACL reconstruction surgery.
2
INTRODUCTION
Approximately 100,000 anterior cruciate ligament (ACL) reconstruction surgeries are
performed each year in the United States, and these operations cost over $2 billion (Gottlob,
Baker, Pellissier, & Colvin, 1999). Despite this high incidence of ACL tears and high cost of
ACL reconstruction, the surgical procedure for repair is far from perfect. Many aspects of ACL
reconstruction, such as graft material, fixation, tension, and flexion angle at the time of
tensioning, fall under the surgeon’s discretion (S. L.-Y. Woo, Wu, Dede, Vercillo, & Noorani,
2006). Therefore, there is no general consensus among doctors or researchers regarding the ideal
ACL reconstruction procedure. As a result, current surgical repair of the ACL is unable to
restore the normal range of movement or curb laxity in the knee (S. L.-Y. Woo, et al., 2006).
The ACL is composed of thick, wavy collagen fiber bundles ensheathed in loose
connective tissue, sometimes showing an alternating fascicular array that is either transverse or
oblique to the long axis of the ligament . Two types of collagen fibrils exist in the ACL. 50.3%
of the fibrils have a non-uniform diameter (25-85nm; peaks at 35, 50, 75 nm) and irregular
outline; where as the other 43.7% are characterized by a uniform diameter (45nm peak) and
smooth margins. Wavy collagen bundles, arranged in various directions, predominate around the
axis of the ACL and the fibroblasts elongate in the direction of these bundles.
(Strocchi,1992). The ACL is structured to withstand multiaxial stresses and tensile strains due to
its varied orientation of bundles, complex ultrastructural organization and abundant elastic
system. Collagen fibril diameter increases with age, leading to an increase in the number of
intermolecular cross-links, which serve to enhance tensile strength. Tight collagen fibrils resist
stretching and smaller, uniform diameter, fibrils are used to resist multidirectional stress
(Strocchi 1992).
3
At present, the factors responsible for the deficiency in biomechanical function in
reconstructed ACL grafts are not well understood. An issue of particular interest in the proposed
research is graft tension. Prior studies have observed a dramatic loss of tensile strength in grafts
post-reconstruction, causing excess knee laxity. In a study of bone-patellar tendon-bone (BPTB)
graft degradation during cyclic loading, graft tension was found to decrease dramatically by 41%
during the first 500 cycles, finally leveling off at 46% after approximately 1500 cycles (Arnold et
al., 2005). This behavior indicates that the initial graft tension at time of surgery is directly
related to knee laxity and therefore is a crucial factor in improving the success rate of ACL
reconstruction (Friederich & O'Brien, 1998).
Determining the extent of tension loss in grafts is essential for standardizing the ACL
reconstruction procedure. However, past studies have limited comparisons to grafts under directloading, cyclic loading outside the reconstructed knee environment, or initial graft tension effects
on clinical outcomes without using imaging to study the effect on morphology (Blythe, Tasker,
& Zioupos, 2006; Gorios et al., 2001) Our study is novel in that it will compare the rate of
tension loss among the BPTB graft, semitendinosus and gracilis hamstring (STG) graft, and ACL
allograft during continuous passive knee motion (CPM).
Varying rates of tension loss may be due to differences in graft collagen organization.
The collagen fibril is the smallest structural unit of tendons and ligaments (Bontempi, 2009).
Generally, these fibrils are linearly arranged and possess a standard crimping pattern along the
longitudinal length of the ligament. In a normal ACL, crimp elongation enables resistance to
higher mechanical stress without permanent deformation (Freeman, 2009). During loading, the
crimped fibrils straighten and then return to their original shapes after stress. Thus, too little
crimp places the graft at greater risk for mechanical damage, and too much crimp may yield
4
excessive joint laxity (Freeman, 2009; Hurschler, Provenzano, & Vanderby, 2003). Differences
in crimp patterns among different graft tissues indicate a direct relationship between the collagen
crimping pattern and the functional mechanical strength of connective tissue (Franchi et al.,
2009).
Thus, we will investigate the correlation between morphological differences, specifically
crimping patterns, in the BPTB, STG, and ACL grafts, with their biomechanical performance
under CPM. We hypothesize that the microstructural organization of collagen is related to the
rates of tension loss among grafts. Collagen organization will be assessed by histological
invasive and non-invasive imaging methods. We expect grafts with more extensive crimping to
maintain tension due to a greater ability to endure mechanical stress. Grafts with excess laxity
after CPM will express a marked difference in morphology, such as an increase in the crimp top
angle and base lengths, indicating crimp elongation.
REVIEW OF LITERATURE
A study by Hadjicostas et al. (2008) compared native ACL tissue, STG, and BPTB grafts
using light microscopy, transmission electron microscopy, immunohistochemistry and
histochemistry. The factors making the native ACL better suited for stabilizing the knee in
comparison to the STG and BPTB grafts were investigated. For example, the concentration of
elastic fibrils was examined, as these structures allow tissues to between withstand multiaxial
stresses and varying tensile strains (Hadjicostas, Soucacos, Koleganova, Krohmer, & Berger,
2008).
Arnold et al. (2005) measured the loss in graft tension over time in BPTB grafts. The
effect of stress on ACL graft integrity and overall knee laxity was investigated, using eight
5
cadaveric knees. Multiple reconstruction surgeries were preformed on the same knee during
testing. A tension transducer was inserted in each graft to measure tension as the knee was
subjected to cyclical flexion-extension while mounted on a motorized rig. The researchers found
that the knees retained approximately half of their initial graft tension after 1500 flexion cycles.
The decrease in tension occurred mostly during the first 100 loading cycles. The drop in graft
tension was found to be directly related to an increase in knee laxity.
Though multiple
reconstructive surgeries were preformed on the same knee, the attachment methods did not
degrade over multiple tests and did not noticeably affect the results (Arnold, et al., 2005).
For the purpose of accurately modeling tension degradation in ACL grafts post
reconstruction surgery, tensile tests should ideally be performed on tendons in reconstructed
human legs. For our research, however, this is not a feasible option. When making
comparisons among graft types it is imperative to minimize confounding variables. One major
variable with this experimental design is that of interspecimen variability. There thus arises the
necessity of performing consecutive reconstruction surgeries on each leg (Savio L. Y. Woo et al.,
2002). In ACL reconstruction surgeries, attachment method is of the most influential variables
affecting graft tension. The most common attachment methods in practice are those of titanium
buttons and interference screws for hamstring and patella grafts respectively (Dargel, Gotter,
Mader, & Penning, 2007). Studies have shown that consecutive reconstructions are possible
when using a titanium button for fixation in the first surgery (Savio L. Y. Woo, et al., 2002).
For our study, however, the focus is on graft type, and the influence of attachment
methods on in situ tensile strength readings must therefore be accounted for. According to Dr.
Leo Pinczewski, titanium buttons and interference screws are highly incongruent. Their
influences in tensile testing would be more significant than any differences that may be seen
6
among graft types. A uniform fixation method must therefore be adopted, and the only viable
option for both graft types is that of interference screws. Unfortunately, inserting such devices is
destructive to the bone tunnels and therefore eliminates the option of performing consecutive
surgeries (D. L. Pinczewski, 2010).
Direct loading of the various tendon grafts is therefore the only remaining option. With
this, it is imperative to obtain a firm grip that is uniform in strength across all samples.
Historically, this has proven to be quite challenging. There exists a very low amount of friction
between clamp materials and wet, soft, collagenous tissues (Cheung & Zhang, 2006). Even
when using high-friction surfaces or serrated jaws, if too little pressure is applied, slippage
commonly results. Conversely, when pressure is too high, the tissue is markedly deformed and
quickly damaged. Further, any form of clamping method creates high stress concentrations at
the edge of the jaws, which may result in premature failure (Cheung & Zhang, 2006; Korvick,
1998; Ramachandran, 2005). Another important factor is that tendons differ in their intrinsic
ability to withstand clamping procedures. The patellar tendon, for example, possesses bone
blocks that allow the clamps to obtain a firm grip on the specimen. Considering that hamstring
grafts do not possess bone blocks, there exists an even greater discrepancy in grip strength
among graft types (Cheung & Zhang, 2006; Korvick, 1998).
The use of cryofixation has been shown to alleviate these attachment issues and is
generally regarded as the gold standard for biomechanical testing of soft tissues (D. L.
Pinczewski, 2010). The two main forms of this attachment method are that of cryofixation and
cryoclamps where the former requires the tissue to be molded into a frozen medium and the latter
uses mechanical clamps to fix onto the frozen end of the tissue (Ramachandran, 2005). These
methods typically use liquid carbon dioxide or nitrogen as the cooling medium (Cheung &
7
Zhang, 2006). They allow for an extremely strong holding connection, which is uniform among
tissue samples. The use of such devices eliminates concerns of stress concentrations, damage to
surface tissue fibers, and slippage, therefore allowing for more reliable data acquisition (Cheung
& Zhang, 2006; Korvick, 1998; Ramachandran, 2005).
A previous study using Sprague-Dawley rats suggested that the structure of tendons and
ligaments are related to their biomechanical properties (Franchi, et al., 2009). The structures of
these tissues include collagen fibrils, which exhibit a sinusoidal pattern known as crimping.
Franchi, et al. (2009) used polarized light microscopy (PLM) to determine crimp number, crimp
top angle, and corresponding crimp base length in tendons and ligaments. Collagen fibrils
change under the stress of mechanical loading. According to Chen, et al. (2007), collagen fibrils
provide resistance to mechanical loads, and a decrease in resistance leads to an impairment in
ligament performance. In a normal ACL, crimping allows the ligament to stretch and to resist
higher mechanical stresses without permanent deformation (Freeman, 2009). During loading,
the crimped fibrils straighten and then return to their original shapes after the stress is removed.
Thus, a small amount of crimping is disadvantageous for the ligament, as it quickens the
progression to plastic deformation.
Excessive amounts of strain may result in permanent
elongation and a perceived loosening of the ligament (Blythe, et al., 2006). By using PLM to
analyze morphological variations in different ACL graft tissues before and after CPM, the
underlying causes of their biomechanical performances can be determined.
According to Fujimoto (2003), optical coherence tomography (OCT) creates an image by
measuring the echo delay and magnitude of light reflected back. Though similar to ultrasound,
OCT has a higher resolution and depth. The image resolution of OCT ranges from 1-15µm:
standard resolution is 10-15µm while laser light sources can provide an image resolution of 1-
8
5µm. OCT generates cross-sectional images of tissue limited to a depth of 2-3mm (Fujimoto,
2003).
A study conducted by Andrews et al. (2008) conducted analyses on OCT images to
measure desired anatomical features. The images revealed the sizes, shapes and thicknesses of
four to five layers of the tubular networks in the kidney’s glomerulus (Andrews et al., 2008). A
follow-up study done by Li et al. (2009) supports that OCT imaging can give significant
information about the histology and pathology of the kidneys. Images were taken at depths of
800μm, allowing visualization of superficial blood vessels (Li et al., 2009). So far, OCT has
been used in ophthalmology as well as in the imaging of arteries and internal organs, such as the
brain and kidney. These studies found that OCT is a useful tool due to its noninvasive nature, the
depth penetration it can achieve, and the availability of 3D imaging in arbitrary planes.
By utilizing OCT imaging, Hansen, Weiss and Barton (2002) studied rat-tail fascicles to
evaluate the feasibility of analyzing crimp patterns and to observe changes in crimping with
applied stress. OCT reflects light off of the crest and trough of each crimp; intervening dark
bands represent the slope of the crimp. The period length of each wave is the width of a band
pair (Hansen, Weiss, & Barton, 2002).
In order to visualize tendon morphology at multiple levels of specificity, OCT will be
used to analyze fascicle crimping in the grafts used in this experiment. Based on the many
studies previously done on OCT, imaging human tissue should be feasible and should provide
good information about the histology of the tissue before and after stress is applied to the graft.
9
MATERIALS AND METHODS
A. Preparation of Specimen
Grafts will be harvested from five pairs of cadaveric knees simultaneously and then
frozen at -20˚C until needed. For this study, a test specimen is defined as a pair of legs
belonging to the same body. Because PLM compromises tissue integrity, it cannot be performed
before mechanical testing.
Since tissues from each leg of the same body should be
morphologically similar, a randomly selected knee will serve as a control and only be used for
preliminary imaging analysis, while the other will undergo ACL reconstruction, mechanical
testing, and post-testing imaging analysis, as shown in Appendix A. A surgeon will harvest the
STG, BPTB and ACL grafts from each leg and perform three separate ACL reconstructions on
each leg with each graft.
Before experimental testing, the legs will be thawed for 24 hours, after which the skin
and soft tissues of the leg will be removed. Osteotomies on the femur and tibia will then be
performed 15cm proximal and distal to the medial joint line, respectively. Capsuloligamentous
structures will be kept to enhance knee stability and allow for more accurate knee motion
(Arnold, et al., 2005).
Figure 1. Diagrams depicting (A) locations of the STG tendons for extraction and (B) the resulting
hamstring graft (L. Pinczewski, Roger, & Scranton Jr., 1998).
For harvesting the STG graft, 10-11cm portions of the tendons will be removed, stripped
of adherent muscle fibers, and trimmed to equal lengths (Figure 1A). These portions will be
10
doubled over and sutured together to form a STG graft, as seen in Figure 1B, with a diameter of
approximately 7-9mm (L. Pinczewski, et al., 1998; Simonian, Cole, &
Bach, 2006; S. L. Y. Woo et al., 2000).
The middle one-third of the patellar tendon, approximately 810mm wide, will be extracted along with bone plugs from the patella
and tibia to form the BPTB graft (Figure 2). Bone plugs should have
the same width as the tendon grafts, ranging anywhere from 9-25mm
long (L. Pinczewski, et al., 1998; Simonian, et al., 2006; S. L. Y. Figure 2. The region of the
BPTB to be excised in graft
harvesting (Pinczewski, et
al., 1998).
Woo, et al., 2000).
B. Pre-CPM Imaging Analysis
PLM and OCT will be performed on the control grafts. Graft tissues to be mechanically
stressed will also undergo OCT because this imaging technique is noninvasive and
nondestructive.
Polarized Light Microscopy
The procedure for analysis of ligament and tendon tissue
by PLM will be performed as directed by Franchi et al. (2008).
The specimens will be prepared in a 10% neutral buffered
formalin solution for 24 hours to initiate fixation.
Before
solidifying the specimens in paraffin, they will be dehydrated with
alcohol.
Then, 10m cross-sections will be stained with 5%
Picrosirius Red and digitally imaged .
Figure 3. Desired measurements
from images taken by PLM.
The collected images will be processed using ImageJ (NIH). Three tissue samples will
be obtained from each graft and ten random images will be taken per tissue. The software will
11
return values for the number of crimps in the sample, the angle of the crimp pattern sinusoid, and
the base- and arc-lengths of each crimp pattern (Figure 3).
Optical Coherence Tomography
The procedure for OCT will be performed as directed by Dr. Yu Chen at the Fischell
Department of Bioengineering, University of Maryland, College Park (2009). In order to obtain
the best 3-D image with improved speed and sensitivity, swept source/Fourier domain (SS-FD)
OCT imaging will be performed with a buffered Fourier domain mode- locking (FDML) laser.
The laser scans 100nm widths at 1300nm wavelengths, thus generating an axial resolution of
10m, or 2.3mm in tissue (Yuan, 2009). The laser sweeps over the tissue at a rate of 16kHz with
an average output power of 12mW. Both the lateral resolutions and magnifications of images
taken can be altered to create optimal images during experimentation. The graft tissue will be
placed on an imaging platform where an infrared FD laser will scan the sample. The penetrating
beam will reflect only a small amount of light back to collecting lens (Yuan, 2009).. OCT will
thus reflect light off the crest and trough of each crimp in the tissue sample; intervening dark
bands represent the slope of the crimp. The period length of each crimp will be the width of a
band pair.
C. Graft Pretensioning Protocol
All grafts should be uniformly pretensioned with 20-80N of force within a range of 0-25º
of knee flexion for 20 minutes.
Though suggestions for amounts of tensioning vary, the
consensus among surgeons is that pretensioning is necessary (Blythe, et al., 2006). Since this
study aims to mimic typical ACL reconstruction as closely as possible, all grafts will be
pretensioned prior to insertion (Arnold, et al., 2005; Dargel, et al., 2007).
12
D. Biomechanical Testing - Continuous Passive Knee Motion
This study chooses to replicate CPM as opposed to cyclical loading. The latter is
intended to model normal walking, but a typical patient uses crutches for a period of at least
three months.
In addition, many patients use a CPM machine during the initial post-
reconstruction period.
After graft harvest, a graft will be mounted onto an MTS machine to replicate CPM. In
order to avoid causing tissue damage to the grafts, cryoclamps will be utilized to achieve a
secure and effect clamping on the MTS machine. The cryoclamps used will be TestResources
G227 Stainless Steel Corrugated Clamps, and will be fitted into the MTS. The test will be
modeled after the OptiFlex® 3 Knee CPM device commonly used for post-reconstruction
rehabilitation ("OptiFlex 3 Knee Continuous Passive Motion," 2009). For this study, the average
patient’s CPM device settings post-reconstruction will be a force of 200N. The knee will
undergo 1500 cycles at a rate of .5 cycles per second ("OptiFlex 3 Knee Continuous Passive
Motion," 2009). While the grafts undergo CPM testing, the MTS will transmit and record realtime data of graft tension and the grafts will be continuously sprayed with saline solution to more
accurately reproduce physiological conditions and prevent dehydration.
E. Post-CPM Imaging Analysis
After CPM, the graft will be removed from the knee and will undergo PLM and OCT
imaging, as described in part B.
F. Statistical Analysis
The MTS will measure loss in tension as a function of the number of cycles. To compare
performance among grafts, an independent two-sample t-test will be applied, assuming an equal
sample size (n=number of cycles) with equal variance. The normality of the data distribution
13
will be confirmed with the Shapiro Wilk Test (Ahmed & McLean, 2002). The Wilcoxon Signed
Rank Test will be used in place of the t-test in case the data is not normally distributed. The
Pearson correlation coefficient will be calculated to determine the dependence of tension loss on
the number of cycles; the coefficient for each graft will be compared to determine which graft
was most affected by CPM (Ahmed & McLean, 2002). After tissue imaging, an ANOVA test
will be used to assess differences among grafts between the before- and after-images of the same
graft (Franchi, et al., 2009).
14
APPENDIX A: EXPERIMENTAL GROUPS
15
APPENDIX B: TIMELINE
16
APPENDIX C: BUDGET
Item
Cost ($)
General
1. Cadaver Legs (10)
2. Transportation
3. Storage and Refrigeration of Legs
4. Laboratory Space
5. Orthopedic Surgeon
Biomechanical Equipment
6. MTS machine (1)
7. G227 Stainless Steel Corrugated
Clamp Grip Bodies (2)
8. G227 Stainless Steel Corrugated
Clamp Jaws (2)
Source
1500.00 Maryland State Anatomy Board
200.00 Based on 15 round trips from College
Park to Baltimore, MD
- Provided by Dr. Adam Hsieh; Lab
Director, Orthopedic Mechanobiology
Lab, University of Maryland
- Provided by Dr. Hsieh
- Recommended by Dr. Hsieh
- Provided by Dr. Hsieh
472.00 TestResources
473.00 TestResources
Tissue Imaging Equipment
9. Polarized Light Microscopy
10. Optical Coherence Tomography
11. Imaging Software, ImageJ
12. Microscopy staining chemicals for
polarized light microscopy
Total
- Provided by Dr. Hsieh
- Provided by Dr. Yu Chen; Assistant
Professor, Optical Imaging, University
of Maryland
- Open source software developed by
NIH
180.00 Fischer Scientific
2825.00
17
APPENDIX C: ACKNOWLEDGMENTS
Team LEGS thanks our mentors, Dr. Adam Hsieh and Hyunchul Kim for all their
wonderful help and advice. We also thank our librarian, Thomas Harrod, for assisting our team
and Dr. Yu Chen for his expert guidance concerning tissue imaging. Finally, we thank Dr.
Rebecca Thomas, Dr. James Wallace, and Courtenay Barrett of the Gemstone Program.
18
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