Histomorphometric Evaluation of Bony healing of Rabbit Critical- sized Calvarial Defects with Hyperbaric Oxygen Therapy A. Jan , G. K. Sàndor, A. Mhawi, S. Peel, C. Clokie, A.W. Evans* (Faculty of Dentistry, University of Toronto, Canada). * Hyperbaric Medicine Unit, Faculty of Medicine, University of Toronto INTRODUCTION A critical sized defect is defined as the smallest full thickness osseous wound that will not heal spontaneously for the life time of the animal. 1,2 Hyperbaric oxygen is being used to facilitate the healing of a variety of hypoxic wounds such as hypoperfused grafts, radiation induced side effects, and necrotizing anaerobic bacterial infections through uprising the oxygen partial pressure in tissues and promoting neovascularization. Muhonen et al 3 conducted ex- vivo studies measuring osteoblastic activity in HBO treated rabbits. The results showed more osteoblastic activity and osteogenic potential in the HBO treated mandibles as compared to the non- HBO group. METHODS 2 groups of 10 New Zealand white rabbits were utilized. All animals had full thickness 15x13 mm (critical-sized) and 18x13 mm (supercritical-sized) calvarial defects created in their parietal bones bilaterally (figure 1). Group 1 (test) received a 90 minutes HBO session at 2.4 ATA per day for 20 days. 3 Group 2 (control) received no HBO therapy. 5 animals were sacrificed at 6 and 12 weeks intervals. Data analysis included qualitative assessment of calvarial specimens and post sacrifice radiographs (figure 2, 3). Thereafter, quantitative histomorphometric analysis was implemented by a blind investigator who analyzed H&E sections (figures 4A,4B). RESULTS Qualitative Findings Gross examination of the calvarial specimens of both groups showed neither signs of inflammation nor hematoma formation in both the 15 mm and the 18 mm defects. All defects showed adequate integrity of the healing wound. Little differences existed in the thickness and density of the defect tissues at 6 weeks and 12 weeks. Radiographs demonstrated more radioopacities in the test group as compared with the control group (figure 3). In the test group, radioopacities observed in margins and in the center of the defects. Histomorphometrics (table 1, figures 4A,4B, 5) Defects in the test group showed more bone formation in comparison with the control group ( p < .001). In the test group, means of new bone formed at 6 and 12 weeks were not significantly different from each other ( p = .309). Furthermore, no significant difference was found in the amount of the new bone formed within the critical and supracritical- sized defects (P = .520). The 12 weeks samples demonstrated more mature lamellar bone, whereas the 6 weeks group showed more woven and less lamellar bone. Figure 1: Bilateral defects in the rabbit parietal bones. Dural blood vessels are evident. Test 15 Test 18 Control 15 Control 18 Test 15 Test 18 Control 15 Control 18 0 10 20 30 40 50 60 70 80 90 100 PERCENT NEW BONE 6 wk 12 wk Figure 4: H&E stained sections (4 X) showing A: A control group specimen healed with fibrous band of tissue. B: HBO treated animal sample showing bony healing of the defect. Figure 3: A radiograph of a test group specimen. Note the relative radioopacity occupying the defect. CONCLUSION This study demonstrates that HBO therapy is an effective measure to enhance bone regeneration in the rabbit calvarial model. The therapy has increased the predicted critical size of rabbit calvarial defects to more than 20%. Hence, HBO therapy could minimize the amount of bone required for grafting. However, further studies are needed to confirm these findings. It is recommended to implement a positive control group such as defects grafted with either autogenous bone or demineralized bone matrix. REFERENCES 1. Clokie CM, Moghadam HG, Jackson MT, Sandor GK. Closure of critical sized defects with allogenic and alloplastic bone substitutes. J Craniofac Surg 2002;13:111-21. 2. Moghadam HG, Sandor GK, Holmes HI, Clokie C. Histomorphometric evaluation of bone regeneration using allogeneic and alloplastic bone substitutes. J Oral Maxillofac Surg 2004;62:202-13. 3. Muhonen A, Haaparanta M, Gronroos T, Bergman J, Knuuti J, Hinkka S, et al. Osteoblastic activity and neoangiogenesis in distracted bone of irradiated rabbit mandible with or without hyperbaric oxygen treatment. Int J Oral Maxillofac Surg 2004;33:173-8. ACKNOWLEDGEMENT This study was supported by Straumann Canada, The Ontario Research and Development Challenge Fund, and The Toronto Academy of Dentistry Figure 5: Percent new bone formation within the defects. Table 1: Percent new bone formation within the defects in the HBO (test) group and non HBO (control) groups OBJECTIVE This study evaluates whether HBO therapy can alter the predicted size of the critical-sized osseous defect in the rabbit calvarial model. Sacrifice Time 6 WEEKS 12 WEEKS Defect Size 15 mm 18 mm 15 mm 18 mm Group Test Control Test Control Test Control Test Control New Bone Formation 64.22 20.63 74.89 30.23 64.02 18.68 58.56 6.84 49.60 22.77 43.36 45.33 58.06 10.07 52.58 20.03 67.22 33.26 75.77 46.55 46.67 14.53 57.35 19.90 53.94 22.64 49.34 24.48 55.39 10.02 50.46 24.16 47.73 20.01 61.04 22.75 56.67 17.61 39.25 42.26 Mean 56.54 23.86 60.88 33.87 56.16 21.46 51.64 26.83 SD 8.74 5.31 14.65 11.37 6.25 18.20 7.67 15.36 SE 3.91 2.41 6.55 5.08 2.80 7.43 3.44 6.27 A B Figure 2: Upon sacrifice, calvarial specimen was dissected free of the cranium. Correspondence: wayne.evans@utoronto.ca