Our Research

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.

Experiment

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).

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
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 8-10mm 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. 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, 10mm cross-sections will be
stained with 5% Picrosirius Red and digitally imaged . 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 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.

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 10mm, 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).

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 real-time data of graft
tension and the grafts will be continuously sprayed with saline solution to more accurately
reproduce physiological conditions and prevent dehydration.