Central defect type partial ACL injury model on goat knees: the effect of infrapatellar fat pad excision

Ten adolescent female Anatolian Black Goats aged between 7 and 11 months were included in the study. Approval and permission from Ege University Animal Care and Use Ethic Committee was obtained for the study. All animals were anesthetized with a ketamine-xylazine combination by a specialist veterinary physician. Five animals were assigned to each of two groups. In group 1, a central defect was created in the ACL of the right knee joints of the animal without any harm to the fat pad tissue. In group 2, the same ACL injury model was performed after complete excision of the infrapatellar fat pad tissue. Left knee joints were left as controls without any intervention.

All knee joints of the animals were prepared under sterile conditions. To decrease intraoperative bleeding and gain clear exposure, subcutaneous jetocaine was injected before the skin incision. A medial parapatellar skin incision approximately 5 cm long was created, and a medial mini-arthrotomy was performed to access the joint in both groups. In group 1, the infrapatellar fat pad and synovial folds were protected and gently retracted to visualize the ACL. A central defect was then created by excising full thickness ligament tissue with a 4-mm arthroscopic punch (Figs. 1 and 2). In group 2, complete excision of the infrapatellar fat pad tissue was performed before creating the same central defect type injury in ACL (Fig. 3). After irrigating the knee joint, the arthrotomy and skin incision were closed in the same manner in both groups.

All animals were restricted to cage activity for 6 weeks after surgery. Analgesia (Metamizole, 20 mg/kg twice daily) and antibiotic prophylaxis (penicillin, 20.000 IU/Kg twice daily) were given during the early postoperative period (48 h). The surgical wounds were closed completely in 10 days. After 6 weeks, the animals were allowed daily activities out of the cages.

Two animals (one from each group) that were chosen at random for histopathology were sacrificed with overdose of pentobarbital at week 10, and the remaining animals were sacrificed at week 12 for mechanical testing. The hind limbs were amputated through the mid-shaft of the femur and tibia and kept at −20 °C until mechanical testing.

Histopathology was performed on the operated and control knees of one randomly selected animal from each group sacrificed during week 10. Macroscopic evaluation was performed to detect the fill of the defect as well as to evaluate for any cartilage lesions. Histological analysis was performed to compare the samples for cellular and vascular responses. Immunohistochemistry was performed to compare the amount of type I collagen at the injury sites.

The samples from the sacrificed animals were stored at 4 °C. The knee joints were carefully dissected. The ACL injury area in the experimental knees and the corresponding area in the control knees were resected and fixed in 10 % formaldehyde solutions for 24 h. Four to five micrometric paraffin blocks were prepared and stained with hematoxylin eosin. Immunohistochemistry assessment for type I collagen was performed in frozen sections taken by cryomicrotome. After an antigen-producing stage with baking (EDTA pH, 8 5 min/850 W microwave), the samples were stained with monoclonal antibody for collagen-1 (collagen 1 antibody; 5D8-G9/Coll-NOVUS) and kept at 4 °C for 24 h. Final labeling was performed with the streptoavidin-biotin method using dab chromogen. Kidney tissue was used for positive comparison.

We measured the structural properties of the ligament as well as sagittal joint laxity (anterior tibial translation) to which the ACL is the primary contributor. A Shimadzu AG 10 K mechanical testing machine was used. In order to attach the joint samples to the machine, a custom device was manufactured as in the previous biomechanical studies [19, 37]. The knee joints of four animals in each group were tested. The previously amputated and frozen limb samples were allowed to thaw at room temperature 12 h before testing. After complete thawing, the soft tissues 5 cm proximal and distal to the joint line were dissected carefully preserving surrounding the soft tissues and joint capsule. The bones were embedded in cylindrical molds with bone cement. To measure anterior tibial translation, the cylindrical molds holding the knee samples were put into the cylindrical parts of the manufactured device and the whole system was located and fixed to the test machine orienting the tibia vertical and the knee flexed to 90°. The joint was cyclically loaded up to 67 Newtons (N) anteriorly and posteriorly at a rate of 20 mm/min (min) in order to simulate the in situ load born by the ACL in goats according to prior work [38]. The mean anterior displacement of 10 cycles of loading was recorded as the mean anterior tibial translation value of the specimens (Fig. 4). After finishing the anterior tibial translation (ATT) test, the specimens were taken out of the system. The remaining soft tissues including the capsule, menisci, collateral ligaments, and posterior cruciate ligament (PCL) were excised to obtain the femur-ACL-tibia complex. The femur-ACL-tibia complex were located and fixed to the test machine again for tensile tests. After pre-tensioning (5 N for 10 min) and pre-conditioning (10 cycles of load up to 20 N) stages, each specimen was tested to failure at 20 mm/min. Ultimate tensile load (Newton), ultimate elongation (mm), stiffness (N/meter), and failure locations were recorded (Fig. 5).

Statistical analysis was performed utilizing SPSSv18 package program. Mann–Whitney U and Wilcoxon’s signed rank test were used for inter- and intragroup analysis, respectively. The level of statistical significance was set at p < 0.05.

Macroscopic evaluations of all knees revealed full filling of injury site (Fig. 6). No cartilage lesion was detected among these samples. Microscopic evaluations showed more fibroblastic cells in the injured ACLs compared to their controls. No inflammatory cells were observed (Table 1). More vascular structures were detected surrounding the defect area in group 2 (fad pad excised). Histological assessment of this group also showed edematous changes and myxoid degeneration in the ligament injury site (Fig. 7). In contrast, normal histological healing of the ACL injury site was observed in group 1 (fat pad protected). More type 1 collagen stained fibers were detected in the injury site of the fat pad excised sample (Table 2).

The study was approved by the Ethic Commission of Ege University Animal Experiments Ethic Commission acceptance number 2010-134.