Construction of the rabbit model of traumatic knee adhesions
Twenty-four New Zealand white rabbits were randomly divided into an experimental group and a control group (
n = 12 in each group). The intervention was performed on the right knee of the rabbits. The rabbits were anesthetized through intravenous injection of 3 % pentobarbitol (1 mL/kg) via the marginal ear vein, and the model was constructed as described by Fukui et al. [
7]. In brief, the hair around the knee joint of the right hind leg was removed using small electric clippers, and the skin was disinfected with povidone iodine. Under sterile conditions, an incision of approximately 3 cm was made lateral to the patella in the right knee on the inner side. The joint capsule was cut open, and the patella was turned outwards. A scalpel blade was used to scratch the synovial membrane on the anterior wall of the suprapatellar bursa, and the scalpel handle was used to push away the attachment between the posterior wall of the suprapatellar bursa and the anterior femur to partially resect the posterior wall of the suprapatellar bursa and infrapatellar fat pad. A sample of tissue was obtained for testing of the baseline collagen content. Haemostasis was performed, and the joint capsule and skin were sutured. Joint puncture was performed to aspirate the intra-articular haematocele, and then HA was injected into the joint cavity.
The rabbits in the experimental and control groups received a 0.3 mL (3 mg) intra-articular injection of HA (Sodium Hyaluronate Injection ARTZ
® Dispo 25 mg/mL, Seikagaku Corp., Japan) or 0.3 mL normal saline, respectively, in the knee joint weekly for 8 weeks. A long-leg circular cast was applied after surgery, and rabbits underwent postoperative external plaster fixation of the right hind limb for 8 weeks with the knee joint extended. A window was opened in the plaster in front of the knee joint so that HA or saline could be injected. The concentration of HA was based on a prior study [
23,
28]. No interventions were performed on the contralateral limb. Rabbits were housed in individual cages after surgery and received intramuscular injection of gentamicin 20,000 units twice per day for 3 days. The cast was removed after 8 weeks, and measurement of range of motion (ROM) of the operated limb was performed. The rabbits were then anesthetized for general observation of the intra-articular adhesions, and synovial tissue on the anterior wall of the suprapatellar bursa was collected to determine the total collagen content.
The ROM of the knee was measured using a plastic goniometer with the rabbit lying on its side at room temperature (22 °C). The goniometer used three points (proximal femur, distal tibia, and lateral femoral condyle) to measure the knee joint angle. The fulcrum was centred over the lateral knee joint space, by palpating the soft-tissue depression just posterior to the lateral tibial plateau. This was the pivot point from which the angle between the tibia and femur was measured. One arm of the goniometer was placed parallel to the line between the greater trochanter and the knee joint, and the other arm was placed parallel to the longitudinal axis of the lower leg and over the ankle at mid-width. Torque of approximately 750–850 g was applied manually to the knee joint during the knee extension measurement, and the distance between the knee joint and the finger placement on the animal’s foot was about 9 cm. An angular velocity of approximately 3°/s was standard. Only one researcher performed the ROM measurement, and he was blinded to group assignment. Three measurements were performed, and the average value was calculated.
Observation of articular adhesions was assessed using Rothkopf’s visual scoring system of bone and joint capsule adhesions [
22] by an experienced pathologist who specialized in joint disease and who was blinded to the study. The degree of adhesion formation was scored: 0, no adhesions; 1, weak, mild adhesions in which the adhesion membrane was thin and could be stripped by minimal manual traction; 2, moderate adhesions which could be removed by manual traction; and 3, dense adhesion layer that could only be removed surgically.
Synovial tissue samples were collected at baseline and after 8 weeks and were stored in a freezer at −20 °C. A 3 mg sample was used for measuring hydroxyproline content using a hydroxyproline kit (Santa Cruz Biotechnology, Santa Cruz, CA, USA) per the manufacturer’s instructions. The total collagen content was calculated based on the fact that collagen contains 13.4 % hydroxyproline, and results were expressed as μg collagen/mg tissue. This study was approved by the institutional review board of Xinqiao Hospital, Clinical Medical Research Center, the Third Military Medical University, Chongqing, China (approval number: xq2011-022), and all animals were treated according to standard guidelines for the ethical treatment and use of animals in research.
Statistical analysis
Eight rabbits, which were not included in this study, were evaluated in a pilot study. It was supposed that the variances of collagen level in the two groups were equal. The results of pilot study showed that the observed variance of the two groups, \( S_{1}^{2} = S_{2}^{2} = 15 \), and the mean difference of collagen, x
1 − x
2, was 3.3. Thus, the sample size in each group should be at least 11 according to the equation \( N = \frac{{t_{\alpha }^{2} \cdot (S_{1}^{2} + S_{2}^{2} )}}{{(x_{1} - x_{2} )^{2} }} \), where α = 0.05 was the significance level. Based on these calculations, we used 24 rabbits, 12 in each group.
Results were presented as mean ± SD, and the differences in ROM, adhesions, and collagen content were assessed by independent two sample t test. The data were normally distributed, and all statistic assessments were evaluated at a two-sided alpha level of 0.05. Statistical analyses were performed using SAS 9.2 statistics software (SAS Institute Inc., Cary, NC, USA).