Preparation of matrices
CollaCote® (Zimmer/dental) is a biodegradable collagen scaffolding used for cellular attachment. Although a collagen sponge has been used clinically as a carrier for rhBMP-2 23, 24), CollaCote® has an allograft matrix possess hemostatic function that facilitates early clot formation and wound stabilization. The final concentration of rhBMP-2 (Peprotech, Rocky Hill, NJ) was dissolved in phosphatebuffered saline (PBS) buffer (pH 7.5) and applied to CollaCote®. CollaCote® was cut with a scalpel into 5 × 20-mm strips and placed with rhBMP-2 in an Eppendorf tube that was left overnight at 4 °C prior to implantation. Similarly, 100 μL of rhBMP-2-free PBS was dropped in CollaCote® to obtain rhBMP-2-free CollaCote®.
Study groups
A total of 52 male Sprague–Dawley rats (8–10 weeks old; CLEA Japan, Inc., Tokyo, Japan) were divided into four groups. Group I (n = 10) included animals that were implanted with control carrier alone. Group II (n = 14) included animals that were implanted with carrier containing 3μg of rhBMP-2. Group III (n = 14) included animals that were implanted with carrier containing 3μg of rhBMP-2, followed by injections of PTH (Teribone; Asahi Kasei Pharma, Tokyo, Japan) (60μg/kg) three times a week (total, 180μg/kg/week). Group IV (n = 14) included animals that were implanted with carrier containing 3 μg of rhBMP-2, followed by injections of PTH (30μg/kg) six times a week (total, 180μg/kg/week). Rats in groups III and IV were subcutaneously injected with PTH beginning 1 week after surgery. The injections were continued until immediately before the rats were euthanized.
Surgical Technique for Constructing the L4-L5 Posterolateral Spinal Fusion Model
All animal studies were approved by the Oita University animal research committee, and experiments conformed to all guidelines and regulations for the protection of welfare of animals (protocol No. 1624002).
The rats were anesthetized by an intraperitoneal injection containing 0.3–0.4 ml of 0.15 mg/kg medetomidine + 2 mg/kg midazolam + 2.5 mg/kg butorphanol. Disinfect the surgical site with 0.05% Chlorhexidine Gluconate. A posterior midline incision was made on the skin. Next, two separate paramedian incisions were made at 3 mm from the midline in the lumbar fascia and the transverse processes were exposed. The transverse processes of L4 and L5 were decorticated using a low-speed burr. Subsequently, CollaCote with or without rhBMP-2 was implanted on each side. The fascial and skin incisions were closed with a 3–0 absorbable suture. After surgery, the rats were given Gentamicin by intramuscular injection for three successive days. Immediately following surgery and on subsequent days, the rodents received analgesics (buprenorphine subcutaneously and paracetamol). The rodents were housed in separate cages and fed food and water ad libitum and their condition was monitored on a daily basis. The rats were humanly euthanized eight weeks post operatively.
Manual assessment of fusion
Eight weeks post-implantation, the explanted spines were manually tested for intersegmental motion by three blinded independent observers 25-31). The explanted lumbar spine was palpated gently and the lateral side bending motion at the L4-L5 level was compared with the motion at the adjacent levels above (L3-L4) and below (L5-L6). The absence of motion was considered as successful fusion. Any motion detected between the transverse processes was considered as a failure of fusion. The spine was designated as “not fused” if any of the three observers graded the spine as not fused. The spines were scored as either fused or not on both the right and left sides and the fusion rate was then calculated.
Radiographic Analysis
The explanted spines obtained at the 8-week time point were photographed using a Softex X-ray apparatus (Softex CSM-2; Softex, Tokyo, Japan) employing HS Fuji Softex film (Fuji Film, Tokyo, Japan) at 45 cm with 30 kV and 15 mA for 20 s. Fusion between the L4 and L5 transverse processes in each rat was recorded as a percentage of the total area between the L4 and L5 that was filled with new bone 25). Three blinded independent observers scored the bone formation in each rat using a 5-point scale: 0 = no bone formation; 1 = bone filling in less than 25% of the area; 2 = bone filling in 25–50% of the area; 3 = bone filling in 50–75% of the area; and 4 = bone filling in 75–100% of the area. The spines were scored on both the right and left sides.
Micro-CT Analysis
The spines were scanned by micro-CT using SkyScan 1172 (Bruker microCT, Kontich, Belgium) with a voxel size of 20 mm. The data were collected at 100 kV and 100 mA and reconstructed using the cone-beam algorithm. Each spine was set on the object stage and sample scanning was performed over a 180˚ rotation with an exposure time of 105 ms. A cylindrical volume of interest with a diameter of 20 mm and a height of 27 mm, which displayed the micro-structure of the rat vertebra as comprised of cortical and cancellous bone, was selected. Data analysis of the area from the top of the L4 transverse processes to the bottom of the L5 transverse processes, including the vertebrae, was performed using the CT Analyzer software (Bruker microCT). The spines were analyzed on both the right and left sides. In the bone mineral density (BMD), tissue volume (TV), bone volume (BV), trabecular thickness (Tb. Th), trabecular spacing (Tb. Sp), and bone volume fraction (BV/TV, %) were measured.
Analysis of Serum Markers of Bone Metabolism
Just prior to euthanization of the animals, blood samples were collected and stored at -80℃ until the serum markers of bone metabolism were analyzed. Serum markers of bone metabolism were analyzed with use of an enzymelinked immunosorbent assay specific for osteocalcin (Osteocalcin High Sensitive EIA kit [rat]; Takara Bio, Shiga, Japan) and tartrate-resistant acid phosphatase-5b (TRACP5b) (RatTRAP Assay; Immunodiagnostic Systems), according to the manufacturer’s instructions.
Histological Analysis
Eight weeks post implantation, the spines were dissected and the specimens were fixed in 40% ethanol, decalcified using standard 10% decalcifying solution HCI (Cal-Ex; Fischer Scientific, Fairlawn, NJ,), washed with running tap water, and then transferred to 75% ethanol. Serial sagittal sections near the transverse processes were cut carefully at the level of the transverse process on both the right and left sides. The specimens were embedded in wax for sectioning. Sagittal sections (5 mm) were cut from the paraffin blocks using a microtome (LS-113; DAIWA-KOKI, Saitama, Japan). The sections were stained with hematoxylin and eosin for basic morphology. Three blinded independent observers scored histological bone formation. Histological fusion was defined as bony trabeculae bridging from one transverse process to the next 25). Fusion masses were assessed and the extent of new bone formation was scored using the following scoring criteria: 1 = fibrocartilage tissue filling less than 25% of the gap area; 2 = fibrocartilage tissue filling 25–50% of the gap area; 3 = fibrocartilage and bone tissue filling 75–99% of the gap area; 4 = bridged with bone tissue, but the fusion masses are comprised of thin trabecular bone; and 5 = completely bridged with abundant mature bone tissue. The spines were scored on both the right and left sides.
Statistical methods
The computer program Statistical Package for the Social Sciences (SPSS) (V13; IBM Corporation, Armonk, NY) was used for statistical analysis. Analysis of variance was used for statistical analysis. p values <0.05 were considered significant. A kappa statistic was calculated as a measure of the interobserver reliability of the three independent blinded observers. The kappa statistic corrects the observed agreement for possible chance agreement among observers. Agreement was rated as follows: poor, k=0–0.20; fair, k=0.21–0.40; moderate, k=0.41–0.60; substantial, k=0.61–0.80; and excellent, k>0.81. A value of one indicated absolute agreement, whereas a value of 0 indicated agreement no better than chance.