We implanted bilateral intercalary allografts and autografts into the right and left ulnas of 13 skeletally mature male coonhounds. Each animal received two allografts, one autograft and one allograft, or two autografts (Table ). Allografts were processed using the standard procedure or the recently developed new process. A 14th animal was used in this study and served as the initial bone graft donor. Power analysis revealed that to detect a mean difference of one in the radiographic scoring of each graft junction, the sample size needed to obtain a power of at least 0.80 (alpha = 0.05) was 20 junctions. The same sample size was needed to determine a minimum difference of 25% in the histologic scoring. We had 13 canines available for this study, with a total of 26 limbs; thus, there were 26 grafts and 52 graft junctions available for review.
The allografts used in this study were prepared by the Musculoskeletal Transplant Foundation (Edison, NJ) staff using the standard or new technique. After sterile recovery of intercalary ulnar grafts from the left and right forelimbs of the initial donor animal, the bone segments were shipped on dry ice to the Musculoskeletal Transplant Foundation facility for final cleaning and processing. One graft was processed using the standard bone graft processing protocol and the other was processed using the sponsor’s new bone graft processing procedure. After completion of processing, the specimens were shipped on dry ice to the study site in sterile packing and ready for surgical implantation. The initial donor animal was used to acquire grafts for the first animal surgery; subsequent surgeries yielded grafts that then were processed and transplanted according to the study design.
The animals fasted overnight before surgery. Thirty minutes before skin incision, 500 mg cefazolin was given intravenously. On the day of surgery, the animals were sedated, intubated, and anesthetized using a combination of thiopental sodium IV(10 mg/kg) and 1% isofluorane as an inhalation anesthetic. Once surgical anesthesia was attained, both forelimbs were shaved and prepared for surgery following standard protocols for asepsis. All surgeries were completed by an orthopaedic surgeon. After the forelimbs were draped, a 10-cm longitudinal skin incision was made centered over the lateral aspect of the ulna. The fascia and underlying tissues were bluntly dissected down to the periosteum of the ulna. Retractors were inserted, and a middiaphyseal 2.5-cm section of bone was removed from the ulna using an air-powered oscillating saw and constant saline irrigation. A standard allograft, new allograft, or autograft was implanted based on the study design. The grafts were tested for fit and adjusted if deemed necessary. The bone grafts were retained in the defect using a seven-hole, 2.7-mm dynamic compression plate (Synthes, Paoli, PA). The three end holes were located in the proximal and distal ends of the resected ulna, while the central hole was located over the graft. The plate and graft were secured using 2.7-mm cortical bone screws at all locations with the graft fixed with standard interfragmentary compression (Fig. ). The tissues then were closed in multiple layers using resorbable sutures and final skin closure achieved using nylon sutures. The surgical site then was covered with a sterile dressing. An identical procedure was performed on the contralateral ulna using the autograft from the contralateral side or allograft according to the study design. The section of removed bone was inserted immediately into a sterile container and placed in an insulated container on dry ice if needed for processing or use as an autograft according to the study design. If the study design required transplantation of an autograft, the opposite surgical limb remained surgically exposed, sterilely, until the autograft could be harvested and transplanted from the contralateral limb.
Surgical placement of a graft is shown.
After completion of the procedure, a 75-μg fentanyl patch was placed and the animals were moved to a special recovery cage where they remained for the first week postsurgery. This cage limits movement of the animal and therefore reduces the risk of perioperative complications. Morphine (0.5 mg/kg) was given IM hourly as needed until 12 hours after fentanyl patch placement. Cefazolin (20 mg/kg) was given twice daily for 3 days postoperatively.
Lateral views of each graft site were taken to confirm graft placement postoperatively and at 90 days to assess displacement of the graft, plate, or screws.
Ninety days after surgery, the animals were euthanized using an intravenous barbiturate overdose in accordance with standard AVMA guidelines. Following euthanasia, the forelimbs were dissected down to reveal the ulna and the graft site. The grafts then were removed by making two cuts in the host’s ulna immediately proximal and distal to the attached bone plate. In this manner, the integrity of the host-graft interface was ensured.
The plates were removed after resection. Using a Hewlett-Packard Faxitron Model 43805 N (Hewlett-Packard, Palo Alto, CA) and Kodak Min-R film (Kodak, Rochester, NY), each specimen was subjected to high-resolution radiographic imaging (Fig. ). These images were used to subjectively evaluate graft integrity and quality of the new bone formed at the graft-host interface. Three blinded observers (JB, RDH, RP) scored the radiographs, using a modified system developed by Stevenson et al. [50
] (Table ). This scoring system rates the distal and proximal unions and appearance and integrity of the host-graft interface, which are combined for a maximum score of eight for each junction. The scores then are added for a maximum score of 16. Interobserver agreement for the radiographic scoring was assessed using Fleiss kappa statistics [57
High-resolution (A) anteroposterior and (B) lateral radiographs show a graft processed with the new cleaning process. This graft received a total radiographic score of 14.7/16.
The retrieved samples were fixed by placing them in 40% ethanol for at least 72 hours. After fixation, each specimen was embedded in polymethylmethacrylate (PMMA) following a standard protocol for large undecalcified bone specimens. After fixing, the samples were dehydrated for a week in graded alcohols to reach 100% ethanol. Once dehydration was complete, the samples were infiltrated in PMMA I, PMMA II, and PMMA III with increasing amounts of catalyst for 5 to 7 days at each step. The samples then were transferred to a final PMMA solution and placed in a water bath (37°C) to polymerize. Once polymerized, two midsagittal wafer sections were cut from each block of tissue. The rough-cut wafer section was mounted on a Plexiglas® slide (Altuglas International, Philadelphia, PA) and ground and polished to a nominal thickness of 75 μm.
Two of the ground and polished slides from each specimen were stained with a combination of Sanderson’s Rapid Bone Stain (Surgipath Medical Industries, Richmond, IL) and Van Gieson’s picrofuchsin (Fig. ). When stained using this protocol, mineralized tissue appears orange to red, collagen fibers green to green-blue, osteoid yellow-green, and muscle fibers blue to blue-green. After staining, the slides were digitally photographed and subjected to histologic image analysis.
This histologic slide shows a graft processed with the new cleaning process. This graft received a 100% histologic union score (Stain, Sanderson’s Rapid Bone Stain and Van Gieson’s picrofuchsin; original magnification, ×7).
We (JB, RDH, RP) used a histologic scoring system, modified from Bos et al. [7
] and developed for this dog model. For a given ulna specimen, two slides were evaluated by three blinded observers (JB, RDH, RP). The proximal and distal graft-host interfaces were evaluated separately. For the proximal interface, cortical bridging was determined on each of the two slides (two cortices per slide, a total of four cortices). The interface received a percent healing score according to the number of cortices bridged by newly formed woven bone: zero cortices bridged = 0%; one cortex bridged = 25%; two cortices bridged = 50%; three cortices bridged = 75%; and four cortices bridged = 100%. The same scheme was used for the distal interfaces. Thus, the scoring system provided for a maximum score of 100% and a minimum score of 0% in four increments of 25% for each graft interface. When evaluating the proximal and distal junctions separately, a histologic score of zero was considered a nonunion. Interobserver agreement for the histologic scoring was assessed using Fleiss kappa statistics.
A histomorphometric evaluation of the host-graft interface was conducted (Image-Pro® Plus; Media Cybernetics, Inc, Bethesda, MD). The areas analyzed spanned a 1-cm segment, 0.5 cm proximal and 0.5 cm distal to each interface. Thus, for each ulna specimen, the proximal and distal graft-host interfaces were histomorphometrically analyzed separately and then jointly. The metrics analyzed were total bone area in the interfacial region and total endosteal/intramedullary bone in the interface region (Figs. , ).
The total interface area measurement is shown.
Endosteal bone area measurement is shown.
Differences in the radiographic scores among the three graft types (allografts processed with the new technique, standard-processed allografts, and autografts) were assessed using the Kruskal-Wallis nonparametric test. Likewise, differences in the subjective histologic scores and histomorphometric scores among the three graft types were assessed using the Kruskal-Wallis test. Means and standard deviations were determined in addition to medians. We used SigmaStat® for Windows (SPSS Inc, Chicago, IL) for all analyses.