All experimental procedures on animals were performed under the guidelines of animal ethics standard regulations approved by the biomedical ethics committee of medical college of Xi’an Jiao Tong University. Twenty-four skeletally mature male New Zealand White rabbits (age, 10–12 months; body weight, 2.9–3.5 kg) were used in this study. All experimental animals were provided by the experimental animal center of Xi’an Jiao Tong University (Production license number SCXK [Shaanxi] 2007-001; the use license SYXK [Shaanxi] 2007-003). All selected experiment animals had no joint swelling, deformity, and limp or abnormal anterior drawer test and Lachman test, and the lateral stress tests were performed on both knee joints and all were negative. Both hind legs were used in each rabbit. On each rabbit, the experimental side leg was randomly selected by coin tossing, and the other side leg was as control.
Rabbits were anesthetized with an intravenous injection of 3.3% pentobarbital sodium (1 mL/kg) (Beijing, China). Both legs were then shaved and aseptically prepared for surgery. The rabbits with obvious joint cavity effusion and synovitis were excluded. On the experimental knee of the animal, an anterolateral skin incision was made and ACL was completely cut off; then, manual examination was used to confirm anterior subluxation of the tibia. After that, the semitendinosus tendon was identified and harvested. Both ends of the tendon were sutured with silk thread and 8-cm-long threads were retained from both graft ends as the traction line; then, the tendon was stretched for 10 min and soaked in physiological saline. According to the diameter of the semitendinosus tendon, a 2.0-mm drill tunnel was made at the intercondylar spine of the tibia and the lateral femoral condyle at the footprints of the normal ACL. Next, the autograft was pulled through the bone tunnels and the graft ends were fixed to the tunnel exits with sutures tied over the neighboring periosteum.
The anterior drawer test and Lachman test were used to check the stability of these knee joints. Then, the joint cavity was flushed, placed with the drainage tube, the wound was closed, and the drainage pipe was connected with a micro-negative pressure aspirator. The negative pressure (50 kPa, 30 min each time, two times a day) was applied after the recovery from anesthesia; the drainage tube was pulled out 5 days after the procedure. The same procedure was applied on the control lower limb but no negative pressure, and the drainage tube was pulled out at the same time with the experimental group. Each rabbit received muscular injection of 800,000 U penicillin sodium for six consecutive days after operation. Postoperatively, the rabbits were returned to their cages and bear full weight. Postprocedural knee joint infection was observed in 1 rabbit, and a total of 23 rabbits (46 knee joints) were involved in the following study. The negative pressure group had a total of 13 left and 10 right knees, on the contrary in the control group.
The rabbits were killed at 6 weeks by air embolism after the procedure. The previous surgical wounds were opened; the knee synovial fluid was obtained and stored at −80 °C. A knee specimen including a 60-mm-long tibia and a 50-mm-long femur was collected from each rabbit. For each specimen, the attached soft tissues were cut off from the graft, suture-tied periosteum also were cut off from outside of the drill tunnel, wrapped in gauze, moistened with physiologic saline solution, and immediately sent for biomechanical testing.
The tibia and the femur were separately embedded in 20 × 20 × 50 mm rectangular aluminum tubes using polymethylmethacrylate resin . The prepared femur–graft–tibia specimens were attached to a microcomputer-controled electronic universal testing machine (WDW-100, Shanghai, China) for tensile test. The loading rate was 5 mm/min and recorded loading for ACL rupture or pulled out from bone tunnels.
Each graft-tibia (or femur) specimen was cut randomly at 5 mm below the tibial tubercle or 1 cm supracondylar femur for histological observation. The samples were sectioned parallel to the longitudinal axis of the tibial (or femur) tunnel. Specimens that the graft pulled out from tunnel were excluded. The specimen was then soaked in a 10% buffered formalin solution. After decalcification, the specimens were sliced through the longitudinal axis of the bone tunnel. After the specimens were being trimmed to 1 cm × 0.5 cm × 0.5 cm sizes, they were cast in a paraffin block and were cut into 5-μm-thick slices; then, they were dewaxed in xylene, rehydrated through a graded ethanol series, washed with phosphate-buffered saline, and stained with hematoxylin and eosin. The histomorphology and vascellum of the tendon–bone interface were observed, and the average number of vascellum was counted with a light microscope for 10 visions of each slice.
Detection of interleukin-1β and tumor necrosis factor-α in synovial fluid
Concentrations of interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) were measured by enzyme linked immunosorbent assay (ELISA), using Quantikine ELISA kits (Shanghai, China).
Immunohistochemical study for vascular endothelial growth factor (VEGF) was carried out by using an avidin–biotinperoxidase complex method and a Histostain SP kit (ZymedLaboratory Inc., San Francisco, CA) according to the manufacturer’s instructions.
Before treatment with the primary antibody, endogenous peroxidase was removed by incubation in 3% H2O2, antigen retrieval was achieved by using trypsin for 5 min, and nonspecific bindings were blocked by treatment with 10% nonimmune normal goat serum at room temperature for 10 min. Human hepatoma tissue was used as a positive control. The secondary antibody of goat anti-rabbit was mixed in incubator temperature for 60 min and was stained with 3,3N-diaminobenzidine. The specimens were then counterstained with hematoxylin.
For each specimen, two blinded pathologists were responsible for counting the total vascular endothelial cells and VEGF-positive in the tendon–bone interface under 10 high-power fields (Olympus; magnification ×400). They then calculated the average number per specimen. Finally, the percentage of VEGF-positive was calculated. Immunoreactivity was graded semi-quantitatively by considering the intensity (0 = negative staining, 1 = light staining, 2 = moderate staining, 3 = strong staining) and percentage of the staining. A histological score was gained from each sample, which was obtained by applying the following formula: total score = (staining intensity × staining percentage)/10.
Statistical analysis was carried out using SPSS17.0 software package (SPSS Co, Chicago, IL). Student’s t test was used to evaluate differences between numeration data. Ranked data was assessed using nonparametric Wilcoxon rank sum test. All statistical tests were two-sided, and P values less than 0.05 were considered statistically significant.