We created a model of OCD in 20 skeletally immature (8-week-old) female New Zealand White rabbits. We first obtained a 4-mm-diameter plug from the weightbearing osteochondral surface harvested on the medial femoral condyle of each knee. We then placed a piece of acellular collagen-glycosaminoglycan matrix into the cavity to replace the plug. Two weeks after surgery, we sedated each rabbit and treated the right knee with ESWT. We used the left knee for a sham-operated control. Multiple radiographs in AP view were taken for both knees. Ten weeks after ESWT, we sacrificed all rabbits and subsequently performed a gross anatomy study and histologic review. The Institutional Animal Care and Use Committee (IACUC) approved this study.
The OCD model in the rabbit knee was as follows. Thirty minutes before surgery, we gave each rabbit a weight-dependent, subcutaneously administered anesthetic mixture of butorphanol (0.5 mg/kg), acepromazine (0.5 mg/kg), and buprenorphine (0.05 mg/kg) to induce anesthesia before surgery. All rabbits were placed on a warm-water-circulating heating pad. During the surgery, each rabbit was administered an inhalation of 2% to 3% isoflurane. We shaved both legs of each rabbit between the upper thigh and upper ankle. The rabbits were placed in a supine position with the knee flexed at 90°. Using a Number 15 scalpel blade, we made an anterior incision approximately 2 cm long on each knee. We cut the skin, fascia, and lateral quadriceps muscle in the proximal to distal direction, and after retraction, the knee was flexed 90°, exposing the medial condyle. We used a 4-mm round dermatologic biopsy punch to make a 4-mm (diameter) by 2-mm (depth) bone and cartilage extraction from the medial femoral condylar region of each knee. Before each cartilage and bone extraction, we made a measured mark on the biopsy punch to maintain depth consistency. A 3- by 3-mm precut piece of Surgisis® Gold Hernia Repair Graft (Cook® Biotech Inc, West Lafayette, IN, USA) was placed into each plug hole and gently pressed into place. Surgisis® is an acellular resorbable Type I collagen-glycosaminoglycan matrix derived from the porcine intestinal submucosa. We then placed the bone and cartilage extraction back into the biopsy cavity, covering the Surgisis® graft. The knee was straightened and a 2–0 Coated VICRYL™ (Polyglactin 910) suture (Ethicon Inc, Somerville, NJ, USA) was used to close the incision. After completion of the operation, we returned all rabbits to their cages and allowed them to move freely. We placed a membrane between the subchondral bone and femoral bone to further delay their union, thereby simulating a typical OCD lesion.
One day after surgery, we subcutaneously injected buprenorphine (0.05 mg/kg) two times daily for 2 days to provide postoperative pain relief. However, one rabbit experienced respiratory arrest. In two rabbits bone and cartilage plugs appeared dislodged on radiographs. We euthanized these three rabbits and removed them from the study. Beuthanasia®-D (1 mL/kg; Intervet/Schering-Plough Animal Health Corp, Summit, NJ, USA) was administered intravenously for euthanasia. Thus, 17 rabbits successfully completed the surgery and were used for the ESWT application. Two weeks after surgery, we anesthetized each rabbit using the methods mentioned above. Fifteen minutes after sedation, we placed each rabbit in a supine position with the right knee flexed at 90°. A coupling gel was applied to the skin surrounding the right knee and the focal point of the OssaTron® (Sanuwave Inc, Alpharetta, GA, USA) shock wave generator was placed 2 to 3 mm from the marked medial edge of the right femoral distal condyle (Fig. ). Each knee was treated with 4000 impulses at a setting of 4 Hz and 18 kV (completed in 17 minutes), corresponding to an energy density of 0.24 mJ/mm2. The left femoral distal condyle on each rabbit was sham treated to mimic the treatment to the right leg and served as the control.
The photograph shows the bone and cartilage plug created using a 4-mm round dermatologic biopsy punch in the rabbit OCD model.
We obtained radiographic films of both the treatment and control groups at 1, 2, 5, and 10 weeks after ESWT. Each rabbit was sedated using weight-dependent subcutaneously administered acepromazine and placed in the supine position, whereupon AP and lateral radiographs of each knee were collected using a GE 2000 x-ray machine (General Electric, Waukesha, WI, USA) with 18- by 24-cm fine-resolution radiograph films. Bone mineral density was calculated for both the treated and control knee using a grayscale level ranging from 0 (white) to 256 (black) on a radiograph film [9
Ten weeks after ESWT, we euthanized all 17 remaining rabbits in the study as described above. After the rabbits were euthanized, we removed both the left and right knees from each rabbit, rinsed them with a solution of 0.9% normal saline, and photographed them with a digital camera. Three observers (MK, XCL, AP) independently examined the control and treated knees and classified the gross morphology of the articular cartilage using a modified Outerbridge Grading System [10
] as follows: Grade 0, tissue completely healed with no evidence of prior injury; Grade 1, tissue displayed softening and swelling; Grade 2, tissue displayed fragmentation and fissuring in a small area; Grade 3, tissue displayed prominent and larger fragmentation and fissuring; and Grade 4, any tissue with major cartilage erosion down to bone. The average agreement among the three reviewers for all cases was 84% (range, 68%–100%).
After completion of gross analysis, we placed each specimen in a 10% zinc formalin solution, removed them after less than 2 hours, and began histologic processing. After 6 hours of decalcification, we collected histologic samples by placing each distal femur into a cutting chamber and performed a sagittal (lateral) cut. The sections we collected were the lateral, middle, and medial regions of the medial condyle. We stained sections using hematoxylin and eosin. Two authors (MK, XCL) independently evaluated each section using the histopathology assessment system [12
] and one pathologist verified the final histologic results. The histopathology assessment ranged from Grades 0 (normal articular cartilage and subchondral bone) to 6 (loss of cartilage tissue and microfracture replaced with fibrocartilage). The average agreement between the two reviewers for all cases was 75% (range, 50%–100%).
Additionally, both bone and cartilage density around the OCD lesion were measured using an Olympus microscope (Olympus America, Inc, Center Valley, PA, USA) and imaging software (MicroSuite™; Olympus America, Inc). Using the Window®
-based software, we identified the region of interests, calculated the density with a grayscale level, and performed statistical analysis at the area. Three coronal sections of the medial femoral condyle with the OCD plug were provided for histologic evaluation. Each slide included the entire bone and cartilage plug and its surrounding area. Three regions of interests were evaluated, including the medial plug border, midplug, and lateral plug border. In each region, the same-sized square was selected and density was calculated. Averaged densities for cartilage and bone were automatically measured for three regions in the computer [23
We performed a Wilcoxon signed-rank test (SPSS®; SPSS Inc, Chicago, IL, USA; MATLAB®; The MathWorks Inc, Natick, MA, USA) to determine differences between the treatment and control knee in the following ordinal variables: gross morphology for the articular cartilage measured by a modified Outerbridge Grading System, radiographic bone density, histologic cartilage and bone density, and histopathology assessment score.