Bilateral femurs and tibiae of 60 sacrificed New Zealand white rabbits (3.5-4 kg, 9-12 months old) were harvested and sent to the Musculoskeletal Transplant Foundation (Edison, NJ, USA), for processing the cortical bone into DBM. The DBM was combined with hyaluronan and porcine gelatin of different jelly strengths to form solid strips. The procedure was identical to that used for processing and production of human DBM, wherein bone harvesting and processing are performed using an aseptic technique.

Posterolateral spinal fusion was performed between the L5 and L6 lumbar vertebrae in 48 skeletally mature (3-3.5 kg, 7-8 month old) New Zealand White rabbits. The rabbits were block randomized into three groups: one control group and two experimental groups. In the control group, 1.4 g of autologous iliac crest bone (group 1) was placed bilaterally. The 1.4 g of bone was weighed to ensure consistency of implant size and then divided into two equal portions for placement bilaterally. In the experimental groups, a high jelly strength DBM-HA-gelatin strip (group 2) and a low jelly strength DBM-HA-gelatin strip (Group 3) were used. The DBM-HA-gelatin strips (25×10×2 mm) were placed directly over the two transverse processes, bilaterally.

A bilateral intertransverse process posterolateral spinal fusion was performed between L5 and L6, similar to that described in previous published studies [91011]. A standard posterior midline incision was used, and bone for grafting was obtained through the same surgical incision. Preoperative antibiotics included 0.25 mL of Dual-cillin (penicillin G benzathine and penicillin G procaine injectable aqueous solution 300,000 u/mL, Phoenix Pharmaceuticals Inc., St. Louis, MO, USA) and 0.25 mL of gentamicin sulfate (Gentocin, 50 mg/mL gentamicin sulfate, Schering-Plough Animal Health Corp., Kenilworth, NJ, USA). General anesthesia was administered by intramuscular injection of a solution (0.75 mL/kg/rabbit) consisting of 150 mg ketamine hydrochloride (Ketaset III, Fort Dodge Animal Health, Fort Dodge, IA, USA), 30 mg xylazine (AnaSed, Lloyd Laboratories, Shenandoah, IA, USA), and 5 mg acepromazine maleate (Boehringer Ingelheim, Vetmedica Inc., St. Joseph, MO, USA). Bupivacaine hydrochloride (5 mL of 0.5%, Abbott Laboratories, North Chicago, IL, USA) was infiltrated intramuscularly into the lumbar paraspinal muscles after a midline skin incision. The transverse processes of L5 and L6 were exposed bilaterally and decorticated with a high-speed carbide burr. Either autologous bone graft or DBM-gel strip was placed over the decorticated transverse processes according to the randomization protocol (Fig. 1). The 1.4 g of bone was weighed to ensure consistency of implant size and then divided into 2 equal portions for placement bilaterally. The surgical sites were closed with running absorbable sutures after hemostasis was ensured. The rabbits were allowed normal cage activity and they were euthanized at 9 weeks post-surgery by intravenous ear vein injection of phenobarbital (2 mL Fatal-Plus solution, Vortech Pharmaceuticals, Dearborn, MI, USA).

After euthanasia, the lumbar vertebrae were dissected with adjacent paravertebral musculature en bloc. A manual manipulation test was performed immediately after harvest, allowing an unencumbered view of the L5-L6 intervertebral disc, and the transverse processes of L5 and L6 [1213]. A reviewer blinded to the group assignment manipulated the spine in the sagittal plane. Only those specimens with complete absence of motion between the transverse processes of L5 and L6 and at the L5-L6 intervertebral disc were rated as fused spines. The specimens were scored as 0 when gross motion was present, 1 if there was limited motion compared with the adjacent segment, or 2 if absolutely no motion was detected.

For radiographic analysis, dorsoventral fine detail radiographs (45 kVps, 1.5 minutes, Faxitron Radiograph Corp., Buffalo Groove, IL, USA) were taken of the specimens. The radiographs were graded in a blinded and independent fashion [9] using a 5-point grading scale.

The volume of the fusion mass was determined from axial computed tomographic (CT) images of the specimen. Two-millimeter images with one-mm overlap (scan circle 150 mm, 200 Mas) were produced with a multi-slice spiral CT (Mx 8000 [quad], Marconi Medical Systems, Cleveland, OH, USA). Each axial CT section was digitized using commercially available software (Software MX-view, Marconi Medical Systems), and the fusion mass area was manually delineated with a cursor. The surface area of all cuts encompassing the spinal fusion was then summated and multiplied by the average cut thickness (one mm), yielding a fusion mass volume [9]. An independent evaluator blinded to the block group randomization of the animals determined the fusion mass volume.

The spines were frozen at -20℃ after manual palpation test and faxitron radiograph. For histologic processing, the spines were subjected to sequential dehydration with ethanol, and then cleared in xylene and embedded in methylmethacrylate. They were cut into sagittal sections approximately 300 µm thick using a water-cooled diamond saw (Isomet, Buehler, Lake Bluff, IL, USA). The sections were mounted on 2-mm thick plastic blocks, carefully ordered. Specimens were ground to 100 µm, polished, and stained with toluidine blue (Fisher Scientific, Fair Lawn, NJ, USA). Selected specimens were subjected to histologic evaluation. Tissues were identified by morphology and by the intensity of the staining to characterize the mineralized tissues.

Statistical analysis was performed using SigmaStat (Jandel Scientific). The presence of bony fusion in the 2 groups, as judged by manual palpation testing and radiographic scores, was determined using the Fisher exact test. For statistical analysis, radiographic grades 3 and 4 were considered fused. The volume of the bone fusion mass in the groups was also compared using the one-way analysis of variance (ANOVA) (Student-Newman-Keuls method for multiple comparisons).