The study design included packing of standard intraosseous defects with bone allografts composed of different particle sizes and quantitating healing of the defects by radiographic, gross, and histomorphometric measurements. Thirty-eight healthy, young and mature outbred male and female baboons (Papio hamadryas) with an average weight of 8.35 kg SD=0.69 were used in the experiments. Skeletal maturity was determined by the closure of the epiphyseal plate in the distal femurs. The animals were kept in colonies in the open–air enclosures. Only one defect per animal was filled with a specific allograft being tested. In total there were 68 defects. Fifty nine were in animals sacrificed at 6 weeks and represented in Table . These were 6 unfilled controls, 6 filled with autograft, 6 filled with 1-2mm cubes, 7 filled with 500-800µm particles, 4 filled with 500-300µm particles 9 filled with 300-90µm particles, 5 filled with 250-125 µm particles, 6 filled with 125-106 µm particles, 4 with 106-75µm particles, and 6 with 75 to 25µm.
Summary of the Statistical Analysis Comparing Individual Experimental Groups (*=P<0.05. NS = not significant)
To determine an appropriate sample size for the study power analysis was performed using Lenth Java applets computer software for power and sample size [19
]. A score of 45, assigned under the scoring system used and derived from comparing control preparations to 1-2mm samples was considered significant, as it indicated some initial bone healing. This value reflects a readily identifiable difference between control preparations that show no healing (assigned score of 0) and the onset of osteogenesis induced by clinically accepted 1-2mm cortical bone. Therefore an effect size of 45 as the standard for significance was set. Assuming a difference in values of the mean = 45 with a standard for significance deviation of 10, to reach statistical significance of p<0.05, a sample of n=4 would give 99.5% power. Using the same assumptions, a minimal sample of 2 would yield a power of 64.6%. Since the intent of the study was to identify bone particles that lead to optimal bone healing, these conditions would reveal effects relevant to outcome.
The animals were fed a standard Purina Monkey Chow (PMI Nutrition International, LLC, Brentwood, MO) diet supplemented with fruit. Before surgical procedures were initiated, the research protocols were approved by Institutional Animal Care and Use Committee. All surgical procedures were carried out in an operation room. Throughout the experiments, the animals were under the care of veterinarians in accordance with USDA regulations and NIH recommendations.
Aseptically excised bone from lower extremities of six animals not included in the group of 38 experimental animals was used for the preparation of allografts. Bone was cleaned of soft tissues, washed with agitation with warm saline to remove bone marrow and fat, wrapped in cotton towels, and sealed in plastic bags. Washing of the graft with removal of bone marrow and extraosseous fat allowed for compacting of the graft material in the defect [11
]. Bone was then rapidly frozen in liquid nitrogen vapor. Aliquots of liquid nitrogen frozen bone were placed into a freeze-dryer chamber using aseptic precautions. The freeze-dryer chambers were sterilized with ethylene oxide and then aerated. Bone was freeze-dried for 5 days with a freeze dryer condenser at-50° to -60°C and shelves at -30° to -20°C. Before removing freeze-dried bone from the apparatus, the shelves were heated to 25°C. The freeze-drying regimen used produced a product with gravimetrically determined residual moisture of 3% to 5%. Freeze-dried bone was ground incrementally in an industrial (Mill- Tek, Tekmar Dohrman, Cincinnati, OH) grinder without overheating. The bone was then sieved through USA Standard Testing sieves. Preparations with particle sizes were produced as follows: 800-500µm, 500-300µm, 300-90µm, 250 to125µm, 125 to 106µm, 106 to 75µm, and 75 to 25µm. These were compared with “crushed bone 1 to 2mm in size. Eighty to 85% of particles in these preparations were within the specified range. The 15% to 20% were smaller.
Animals immobilized with ketamine (5 mg/kg of body weight) were intubated and anesthetized with isoflurane. Vital signs were monitored throughout aseptically performed surgical procedures. Preoperatively, animals were given 25 mg/kg cefazolin. Cefazolin was also administered twice a day for 3 days postsurgery. After the operation, animals were maintained individually in cages for 3 to 4 days. Analgesia was provided with burprenorphine (0.01 mg/kg of body weight) every 12 hours for 1 to 2 days. Animals were then returned to their respective colonies. Food and water was given ad libitum.
Distal femurs and proximal tibias were approached through anterior incisions. Corticomedullary defects measuring 9 to 10mm in diameter and 15mm in depth were created with intermittent burring with saline irrigation in the metaphysis- diaphysis of the distal femur and the proximal tibia. Sometimes the defects involved the epiphyseal line. The defects extended into medullary canal. Consequently, there was considerable bleeding. Hemorrhage was controlled by packing the cavity with bone graft which acted as an excellent hemostatic agent. Control (unpacked) defects were packed with gauze sponge which was left in place under pressure until the bleeding subsided. One or two defects were placed in the distal femur and the proximal tibia in each animal. In animals with small tibias no defects were made in the same tibia because of fear of fracture. For the present study fifty nine defects were made. Only one defect per animal was filled with a particular type of allograft under study. Autologous bone was bone slurry obtained from burring of defects. Since only one specific type of allograft was implanted per animal each defect was considered an independent sample. The technique of creating and filing the defects with preparations under study consisted of burring holes with frequent irrigation with saline, measuring the depth of the defect, filling it with particulate allograft and tampering the same. Unfilled defects served as negative controls and those filled with autografts obtained with burr from two or more sites were positive controls. Roentgenograms (anteroposterior and lateral) were obtained postoperatively and at 6 weeks. Twenty nine of 38 animals were sacrificed at 6 weeks and 9 at 12 weeks. Euthanasia was achieved by an intravenous injection of FATAL-PLUS (Vortech Pharmaceutical, Dearborn, Michigan). Before sacrifice at 6 weeks limbs of experimental animals were radiographed. Defects with smaller particles (75-25µ) were not healed at this time. Therefore a decision was made to maintain representative animals for total of 12 weeks to determine if these would heal by that time. These included 2 animals each with 75-25 µm and 300-90 µm particles, one animal each with 1-2mm, 800-500 µm and 500-300 µm particles and 2 controls. Femurs and tibias were dissected and frozen in dry ice. The bones were then sectioned with a diamond saw, photographed, and radiographed. Bone specimens were fixed in 10% formalin- Earle’s balanced salt solution mixture, decalcified in Perenyi’s fluid (10% nitric acid and 0.15% chromic acid in 30% alcohol), embedded in paraffin, and sectioned at 5 to 7µm. Sections were stained with hematoxylin and eosin, PAS-hematoxylin, Romanowski-Giemsa, and Masson’s trichrome stains. The results were expressed as a combination of gross, radiographic and histomorphometric data. Results were averaged for each experimental group. A total score of 100 was assigned to bone which was normal grossly, radiographically, and microscopically. A score of 0 would be assigned to an unaltered defect if the cavity was completely unhealed and remained open (Table ).
Method of Grading Healing of Bone Defects
Histomorphometric measurements were based on color conversion generated by translating grey scale image to 20 color representations to visualize distinct differences which correlated with histologically apparent new bone formation using NIH image 1.62. Area within a defect occupied by newly formed bone was measured using Image ProPlus
Version 5.0 for Windows. The grading was performed by two authors(TM and HTT) on coded specimens. It is of course, obvious that specimens with large particles could not be mistaken for those with smaller particles. However, specimens with particles 506-300µm, 300-90µm-250-125µm and 125-106µm had similar appearance. Interobserver variability was within 15-20%. In arriving at a final score the differences were averaged. Data from the scores for experimental and control groups, as previously defined, were compared with each other using a one-tailed Student’s t-test assuming two-sample unequal variance. Value of p<0.05 was considered significant.