This study was performed in accordance with Institutional and National Institutes of Health regulations governing the treatment of vertebrate animals. It was initiated after approval by the University of Wisconsin-Madison Animal Care Committee.
Ten skeletally mature female crossbreed dogs, weighing 24–37 kg each (27 ± 1.3 kg) (mean ± SEM) , were used in this study. MRFE was applied to the ACL of one randomly selected knee joint of each dog arthroscopically. An identical operation was performed o n the contralateral joint with the exception that no energy was applied to the ligament. Treated ACLs ruptured approximately 53 days after surgery. Animals were sacrificed 16 weeks (n=5) or 26 weeks (n=5) (Beuthanasia-D Special, Schering-Plough Animal Health, Union, NJ, USA) after rupture of the treated ACL, and the hind limbs were stored at -70 °C until biomechanical testing. Comparisons were made between knees with ruptured ACLs with and without JC and control joints with and without JC. Assessment parameters included manual knee examination, radiographs, gait analysis, and instrumented anterior-posterior (AP) translation and medial-lateral (ML) rotation testing. Synovial fluid levels of the OA markers 3B3(-) (proteoglycan) and COL2-3/4C long (collagen type 11) were determined after each radiograph, and are reported elsewhere [2
]. Open dissection confirmed that all treated ACLs were ruptured.
All animals were pre-medicated with a subcutaneous injection of 0.10 mg/kg acepromazine (Acepromazine maleate, Vedco, St. Joseph, M O USA) and 0.2 mg/kg butorphanol (Torbugesic, Fort Dodge Animal Health, Fort Dodge, IA, USA). After 20 min, 5 mg/kg thiopental (Pentothal, Abbott Laboratories, North Chicago, IL, USA) was administered intravenously for induction of anesthesia. The dogs were intubated and maintained o n halothane in oxygen administered with a semi-closed circle system. Each animal received an intravenous preoperative injection of cephazolin (20 mg/kg) (Cephazolin, Faulding Pharmaceutical Co., Elizabeth, NJ, USA) for prophylaxis. Each knee was physically examined for drawer, range of motion, swelling, temperature, crepitus, patellar tracking, and varus-valgus. A standard surgical scrub with chlorhexidine followed by a wash with sterile water was performed.
The knee to receive treatment was distended with 7 ml of sterile saline. A 0.5 cm incision was made through skin and J C on the lateral aspect of the knee 0.5 cm proximal to the tibia and just cranial to the lateral digital extensor tendon. A 2.7 mm blunt trocar in a 3 mm cannula was used to enter the joint through the incision. The trocar was replaced by a 2.7 mm, 30° arthroscope (Storz, Goleta, CA). Joint distension was maintained with sterile saline using gravity flow. A 4.0 mm synovial shaver (APEX, Linvatec, Largo, FL) was placed in the joint through a 0.5 cm incision in the medial aspect of the joint, just proximal to the tibial plateau and just medial to the patellar tendon. Synovium and infrapatellar fat pad were removed until the origin and insertion of the ACL could be clearly seen. A 2 mm diameter MRFE probe (Tac-C probe, Smith & Nephew, Inc., Andover, MA) was used to treat the entire anterior surface of the ACL in transverse passes across the ligament at a rate of 1–2 m d s at temperature and power settings of 70 °C and 25 W, respectively, using a MRFE generator (Electrothermal System ORA-50TM, Smith & Nephew, Inc., Andover, MA). The joint was lavaged for 1 min with sterile saline, and incisions were closed routinely. An identical operation was performed on the contralateral joint except that n o energy was applied to the ACL.
Force plate gait analysis was performed pre-operatively and at 4, 8, 12, 16, and 24 weeks after surgery for all dogs, and at 34 weeks after surgery for five dogs using a force plate (OR6-6-1000 Biomechanics Platform with SGA6–4 Signal Conditioner/Amplifier, Advanced Medical Technology, Inc., Newton, MA) connected to a commercially available satellite data acquisition system (VETDATA v2.03, Acquire v5.0, Mininet v4.0 and Update vl.1 from Sharon Software Inc., Dewitt, MI). A single handler trotted dogs for all gait trials. A trial was considered successful if a forepaw contacted the force plate followed by contact of the ipsilateral hind paw at a velocity of 1.80–2.80 m/s and acceleration of 0.9 to–0.9 m/ s / s . The velocity range was designed to include a comfortable trot rate of each dog included in the study. Three successful passes for each limb were recorded at each time point. Peak vertical impulse (PVI) values, defined as total force applied over time, were normalized to body weight for comparison across time.
Radiographs (posteroanterior and lateromedial) were taken pre- operatively and at 4, 8, 12, 16, and 24 weeks after surgery for all dogs, and at 34 weeks after surgery for five dogs. All radiographs were performed with the knees in full extension (approximately 30° of flexion). Radiographs were evaluated for signs of OA including the presence of osteophytes, enthesiophytes, subchondral bone sclerosis, and, following ACL rupture, anterior displacement of the tibia. All radiographs were performed with the animals sedated (acepromazine, 0.10 mg/kg; butorphanol. 0.2 mg/kg; both administered subcutaneously).
A P translation and ML rotation were performed with the knee at 30°, 60°, and 90° of flexion. Each A P translation test was followed by a ML rotation test before the joint angle was changed. All testing was first done with an intact JC and then with J C dissected away. JC was always completely detached from all femoral and tibial insertions, though small pieces were left attached to periarticular structures as necessary to ensure that the structures were not disrupted. The quadriceps muscle was transected 6 cm proximal to the knee for all testing, and the quddriceps, patella, and associated tendons were reflected in the anterior direction when JC was removed.
The tibia and femur were cut to a length of 10 cm from the joint line and were potted with polyester resin in thick-walled aluminum cylinders for attachment to mechanical testing system (MTS 858, Minneapolis, MN). Musculature was removed 6 cm above and below the joint line. The specimen was then mounted in the testing system with the tibial cylinder rigidly fixed to a base attached to the force-moment sensor and the femoral cylinder rigidly fixed to the actuator in custom made positioning fixtures (). The positioning fixtures permitted placement of the knee in three different flexion angles, 30°, 60°, and 90°. It accommodated six degrees of freedom for positional adjustment. The degrees of freedom were referenced to the anatomic planes of motion: AP, ML, and superior-inferior displacement, flexion-extension, varus- valgus, and internakxternal rotation. During adjustment of knee flexion angle, internal-external rotation of the tibia was unlocked and free to rotate. At the desired flexion angle, the tibia was rotated internally and externally until the midpoint of rotation was established, and the tibia was locked into position. Internal-external rotation of the tibia relative to the femur was locked while A P loads were applied across the tibiofemoral joint t o yield reproducible load-displacement data.
Fig. 1 The knee-testing fixture allowed six degrees of freedom to position the joint. The rotational axes of the fixture were aligned with the rotational axes of the canine knee. Load (AP or ML) was applied to the femur through the Materials Test System while (more ...)
A P translation was evaluated by applying four complete AP load cycles to each specimen. The first two load cycles were used to pre-condition the specimen and to demonstrate reproducible load-displacement of the tibia relative to the femur that occurred between the limits of the 20 N anterior and 20 N posterior loads. The joints were then tested non-destructively through two cycles under load control (ramped over 12 s per cycle). AP knee translation was defined as the AP displacement of the tibia relative to the femur that occurred between the limits of the 40 N anterior and 40 N posterior loads.
Following each AP translation test, a ML rotation test was performed before the angle of the joint was changed for the next test. For this study, ML rotation refers to internal (medial) rotation of the femur relative to the tibia followed by lateral (external) rotation of the femur relative to the tibia. The joints were tested non-destructively through four cycles as described previously. For pre-conditioning, two cycles of 2 N m were applied to the femur first in the medial direction followed by the lateral direction. For testing, a torque of 4 N m was applied to the femur medial to lateral in two cycles (ramped over 12 s per cycle). ML rotation was defined as the ML rotation of the tibia relative to the femur that occurred between the limits of the 4 N m medial and lateral loads.
Comparisons in AP translation and ML rotation were made among knees with ACL rupture with and without JC and control joints with and without JC. The SAS least squares mean and SEM was determined for each property of interest. A factorial model with all interactions was used to compare time, capsule status, ligament status, and angle. Because all treatments except time were within-animal treatments, a split plot model for incomplete data was used. Standard errors for the means were based on the root means squared error from the ANOVA. The mean and SEM for PVI, velocity, and acceleration were determined for each treatment group at all time points. ANOVA was used to determine differences in velocity or acceleration among trials (treated and sham treated ACLs at each time interval) and among time intervals for PVI of each treatment group. Tukey’s post-hoc analysis was used to evaluate significant differences among time intervals. Student’s t-tests were used to determine differences in PVI among treatment groups at each time interval. Significance for all analyses was set at P < 0.05. Statistical analysis was performed with commercially available software programs (SAS Release 6.12, SAS Institute, Cary, NC, USA or GraphPad Prism v3.0, GraphPad Software, Inc., San Diego, CA, USA).