Animals and Groups
Eighteen healthy and skeletal mature beagles (19 months old on average), weight 16 to 21 kg and average weight 18kg, were used in this study. The experimental animals were provided by the animal experimental center of the Shanghai Jiao Tong University Affiliated Sixth People’s Hospital. And experiments of animals were approved by the Animal Research Committee of Sixth People’s Hospital, Shanghai Jiao Tong University School of Medicine. All animals were healthy and free of infection, and were magnetic resonance imaging (MRI) scanned of the spine to assure the absence of Intervertebral disc (IVD) degeneration related diseases before the study. 18 beagles were bred and randomly allocated into the following study groups:(1)L4-5 lumbar interbody fusion（IF）. (2) L4-5 lumbar interbody fusion +L5-6 interspinous Coflex implantation（Cof1）.(3) L4-5 and L6-7 interbody fusion+ L5-6 interspinous Coflex implantation （Cof2）. In all animals, L5-6 discs were punctured to generate degeneration ，and the intact L2–3 disc served as a noninjuries control (Con group). The animals were followed up for 6 months after the initial operation. The changes in the L5/L6 lumbar discs were assessed using gross anatomical observation, MRI, histology, and gene expression analysis at 3 and 6 months respectively. At each point（3 months and 6 months postoperatively）, 3 beagles were randomly selected from each group to be killed with an excess dose of ketamine hydrochloride and xylazine hydrochloride injection after MRI scan.
Preparation of Autologous Iliac Bone Graft
24 hours abstinence for food and 12 hours abstinence for water was maintained before surgery, and Surgical procedures were performed under general anesthesia by intramuscular administration of ketamine hydrochloride injection (0.1 ml/kg) and xylazine hydrochloride (0.08 ml/kg). After anesthetization, the dogs were placed in the lateral position. The incision was 4 cm along the lateral border of the spina iliac anterior superior. After exposure of spina iliac anterior superior, an approximately 3 × 2-cm autologous iliac bone graft was obtained by bone drill. The autologous iliac bone graft was prepared for anterior lumbar interbody fusion.
Anterior lumbar interbody fusion and implantation of Coflex
When the harvesting of iliac bone graft finished, beagles for experiment were placed in a supine position, median abdominal incision were delivered, then lateral incision approach of the rectus abdominis was achieved, once an opening through the muscle was obtained, carefully protect the peritoneum and reflect it anteriorly by blunt dissection. After retraction of the peritoneum and its contents using a wide retractor, the appropriate involved vertebras were identified. Separate the intervertebral disc and annulus from the cartilaginous endplates of the vertebrae with a thin osteotome, and then the disc was removed by pituitary rongeurs, Kerrison rangers and curets. When interbody space was cleaned and prepared for fusion, predisposed grafts were used to complete interbody fusion. After completion of the fusion, close all layers with absorbable sutures. When the interbody fusion was delivered, the Coflex groups underwent additional implantation of the Coflex (Paradigm Spine, LCC, New York) at the involved level. The beagles were placed in prone position, surgery was performed through a standard posterior midline approach, and then the interspinous ligament was dissected and excised. The device was inserted between the adjacent spinous processes and the flanges were crimped follow the manufacturer’s instructions, so that it was seated fitly. Appropriate placement of the implant and adequate segmental sagittal alignment were identified under C-arm machine.
Generation of L5-6 disc degeneration
After all of surgical procedures described above were done, the beagles were placed in prone position, L5–6 discs were then punctured with 18-gauge needles（HuaYi Bio-technology, Shanghai, China） along the outside of the facet joints under C-arm fluoroscopic guidance until the needle tips reached contralateral side of the discs both in anterior-posterior and lateral radiographs. Following the operation, antibiotic was intravenously delivered in five consecutive days. Beagles were fed in cages, and food and water were placed at a relatively high place, which would force them in erect position more than 8 hours per day. The animals were monitored daily for potential complications or abnormal behavior. Important surgical procedures were shown in Fig. 1.
Magnetic resonance imaging
MRI (Achieva 3.0T. Philips, Holland) scans were administered to evaluate signal changes in T2-weighted (T2-W; TR 2270 ms, TE 126 ms) images at baseline, 3 and 6 months after the operation in all groups. The subjects were imaged at the time of follow-up using the same scanner and examination protocol, and the slice thickness and interslice gap were 4 mm and 0.4 mm for sagittal. Lumbar IVD degeneration was graded on T2-weighted MR images according to the Pfirrmann classification system on a scale of I–V, where grade V denotes the most degenerate category . All radiological assessments were made independently by two observers, including one radiologist and one orthopedic spine surgeon. when disagreement occurred with respect to the radiological grade, a consensus opinion with involvement of a third observer was sought.
Gross anatomical observation
At 3 and 6 months, after MRI scan, 3 beagles of each groups were killed with an excess dose of ketamine hydrochloride and xylazine hydrochloride injection, and the spines were harvested. Then the L2-3 and L5–6 discs were isolated intact, bilateral cartilage endplate and some vertebral body were preserved. The discs from each dog were cut coronally at the center of the disc for Gross anatomical observation.
Histological and immunohistochemically analysis
All L5-6 discs of 18 dogs were cut transversally at the center of the nucleus pulposus (NP). One half of every disc was used for histological studies, and the other half was used for Realtime PCR analysis of gene expression. The NP tissues were isolated immediately, and fixation were done in 10 % neutral-buffered formalin for 72 hours and then processed for paraffin embedding and cut into transversal sections (6 μm thick) using a microtome. The sections were stained with hematoxylin and eosin for evaluation. Immunohistochemical detection of Col I, Col II performed using formalin fixed sections obtained as described above. Briefly, NP tissue sections were maintained at room temperature for 60 minutes and dewaxed by xylene twice (10 minutes each time). The tissues were then rehydrated by a series of 5-minute washed in 100 %, 95 %, 80 %, and 70 % ethanol, followed by 5-minute washed in distilled water and three consecutive 3-minute washed with PBS (PBS; Gibco Grand Island, New York, USA). Incubation in 3 % hydrogen peroxide for 10 minutes were delivered in purpose of inactivating the endogenous peroxidase, after that, antigen retrieval was performed by heating the samples at 95 °C for 20 minutes in 10 mM sodium citrate (pH 6.0). Nonspecific binding was blocked by incubating with 10 % normal goat serum (Gibco Grand Island, New York, USA) for 20 minutes, then the NP tissue sections were labeled overnight at 4 °C with primary antibody: anti-Col I (1:200dilution), anti-Col II (1:200 dilution), polyclonal antibodies (immunoglobulin G) (Hua An Biotech, Hangzhou , China). The NP sections were then incubated for 60 minutes each with a horseradish peroxidase-labeled secondary antibody and then streptavidin–peroxidase (Hua An Biotech). After washing with PBS, the sections were incubated with 3,30-diaminobenzidine substrate until a brown color generated. Finally, the sections were counterstained with hematoxylin. Dehydration was performed by using a series of 2-minute washes in 50 %, 70 %, 95 %, 95 %, and 100 % ethanol. After two consequent 2-minute washes with xylene, the slides were sealed with coverslips. To quantify the immunohistochemical results, staining intensity was analyzed using the Image-Pro Plus 6.0 (Media Cybernetics, Rockville, Maryland, USA). The area of interest in all sections was analyzed, and the mean density was calculated by integrated optical density divided by the area.
Real-time PCR analysis of gene expression
Total RNA was extracted from the NP using the Trizol reagent (Life Technologies) according to the manufacturer’s instructions. RNA was reverse transcribed into cDNA using AMV reverse transcriptase (Life Technologies). After the cDNA had been obtained by reverse transcription, relative gene expressions of COL1, COL2, TIMP-1 and BMP2 were determined by real-time PCR and normalized to the glyceraldehyde-3-phosphate dehydrogenase housekeeping gene. These primers were designed using Primer Premier 6.0 software (PREMIER Biosoft, palo alto, California, USA) (Table 1). The Mini Opticon™ Detector System (Life Technologies) and the SYBR Green PCR kit (Life Technologies) were used for Realtime PCR analysis. The real-time PCR consisted of an initial enzyme activation step at 95 °C for 20 seconds, followed by 40 cycles of 95 °C for 5 seconds and 60 °C for 20 seconds. A cycle threshold (Ct) value was obtained for each sample, and triplicate sample values were averaged. The 2–ΔΔCt value was then used to calculate relative expression of each target gene . The data presented (mean) were from three independent experiments in which both sample sets were analyzed in triplicate.
All data were statistically analyzed with GraphPad Prism (v6.0). Error bars in graphical data represent mean ±s.d. Statistical significance was determined using a Mann–Whitney U test, in which p values of p ＜0.05 were considered statistically significant. Variance was similar between the groups that were statistically compared.