Zentrum für Translationale Knochen-, Gelenk- und Weichgewebeforschung Establishment of a femoral bone defect model for immunodeficient mice S. Zwingenberger 1,4 , E. Niederlohmann 1 , C. Vater 1 , S. Rammelt 1 , R. Matthes 2 , R. Bernhardt 3 , S. Kirschner 1 , R.D. Valladares 4 , S.B. Goodman 4 , M. Stiehler 1 1 University Hospital Carl Gustav Carus at Technical University Dresden, Center for Translational Bone, Joint and Soft Tissue Research, Germany 2 AO Research Institute Davos, Switzerland 3 Max Bergmann Center for Biomaterials Dresden, Germany 4 Department of Orthopaedic Surgery, Stanford University, USA Background Background Background Background In order to develop effective therapies for bone regeneration, site-specific bone defect small animal models are frequently needed. However, publications on murine bone fixation devices suitable for the generation of critical-size segmental bone defects are sparse. Therefore, the aim of this study was to establish a reliable, easy-to-handle, critical-size bone defect model using immunodeficient mice. Results Results Results Results All of the animals survived the operation. 6 out of 30 animals (n=2 per each group) died before the end of the observation period due to extended blood loss (n=1), infection (n=1), euthanization due to pin loosening (n=2) or anaesthetic side effects during X-ray follow up (n=2). The postoperative X-rays up until 12 weeks (Figure 6) showed a reduction of the gap size from 1.19±0,25 mm to 0±0 mm (1 mm group), 2.09±0.30 mm to 0.29±0.38 mm (2 mm group) and 2.98±0.18 mm to 0.77±0.56 mm (3 mm group). µCT analysis demonstrated that after 12 weeks (Figures 7 and 8) the defect volume was significantly different between the 3 groups (p<0.001). The 1 mm group reached a defect volume of 0.36±0,42 mm³, 2 mm group 1.40±0.88 mm³ and 3 mm group 2.88±0,28 mm³. The percent reduction of the defect size during the 12 week postoperative period (Figure 9, p=0.152) was also significantly different between the groups. The defect size decreased by 77.6% (1 mm group), 56.8% (2 mm group) and 28.6% (3 mm group). To evaluate bone healing histologically we used a scoring system developed by Huo et al. 1 (Table 1, Figures 10 and 11). The 1 mm group had a median score of 8, meaning that the former defect gap was filled predominantly by immature bone with a small amount of cartilage. In the 2mm group we found a score of 4,5 and in the 3mm group the score was 2.5, meaning that the former defect gap was filled predominantly with fibrous tissue and with a small amount of cartilaginous tissue. There was a local statistical significance between the 1 mm group and the 3 mm group (p=0.038). For the histomorphometric analysis we stained for osteoclast-specific TRAP (tartrate-resistant acid phosphatase) and evaluated the slides using an optical magnification of 25 fold with a standard 10mm x 10mm grid (Figure 12). For the 1 mm defects we counted 1 grid, 2 grids for the 2 mm defects, and 3 grids for the 3mm defects. The groups had an average of 3,3 cells, 2,8 cells, and 2,6 cells per field for the 1, 2, and 3 mm groups, respectively. Figure 1 Figure 1 Figure 1 Figure 1: External fixateur used Contact: Contact: Contact: Contact: Klinik und Poliklinik für Orthopädie Universitätsklinikum Carl Gustav Carus an der Technischen Universität Dresden Dr. med. Stefan Zwingenberger http://ortho.uniklinikum-dresden.de Stefan.Zwingenberger@uniklinikum-dresden.de Methods Methods Methods Methods Thirty male nu/nu nude mice (40.7 ± 2.8 g, 95 ± 2.6 days old) were anesthetized by isoflurane inhalation and an external fixation device (MouseExFix, RISystem, AO Research Institute Davos, Switzerland) was attached to the right femur. Femoral bone defects of 1 mm (n=10), 2 mm (n=10), or 3 mm (n=10) were created using a gigli saw. Wounds were closed with interrupted sutures. X-ray films were obtained immediately upon surgery and every 2 weeks postoperatively. Postoperative observation time was 12 weeks. After 12 weeks the animals were euthanized and histomorphology, histomorphometry, immunohistochemistry, and µCT analysis were performed on the femurs. Conclusion Conclusion Conclusion Conclusion The mouse ExFix device is a reliable and easy-to-handle system for the creation of critical-size segmental bone defects in immunodeficient mice when 3 mm defects are generated. This mouse model allows for high-throughput translational evaluation of concepts for site-specific bone regeneration including strategies using allogenic and xenogenic cell types. Acknowledgements Acknowledgements Acknowledgements Acknowledgements This work was supported by the AO Foundation (Start up Grant S-10- 67) and German Academic Exchange Service/Federal Ministry of Education and Research (grant # D/09/04774). Figure Figure Figure Figure 2: Creation of the bone defect using a spacer and a gigli wire Figure Figure Figure Figure 3: 3: 3: 3: Postoperative X-ray of a 1 mm defect Figure Figure Figure Figure 4: Postoperative X-ray of a 2 mm defect Table 1 Table 1 Table 1 Table 1: Histological scoring system according to Huo et al. 1 Figure Figure Figure Figure 10 10 10 10: Histological section of an initial 3mm defect showing a non-union, Huo Score 2, H&E stained, 25X Figure Figure Figure Figure 12 12 12 12: Histomorphometrical analysis of TRAP stained osteoblasts per field References: References: References: References: 1) Huo MH, Troiano NW, Pelker RR, Gundberg CM, Friedlaender GE. The Influence of Ibuprofen on Fracture Repair: Biomechanical, Biochemical, Histologic, and Histomorphometric Parameters in Rats. Journal of Orthopaedic Research. 1991; 9: 383-390 Figure Figure Figure Figure 5: Postoperative X-ray of a 3 mm defect defect volumes after 12 weeks 1 mm 2 mm 3 mm 0 1 2 3 *p<0.001 group * * * Figure Figure Figure Figure 6: defect size progress throughout 12 weeks as evaluated by X-ray Figure Figure Figure Figure 7: defect volumes after 12 weeks as evaluated by µCT Score Score Score Score Histological Findings Histological Findings Histological Findings Histological Findings 1 Fibrous tissue 2 Predominantly fibrous tissue with small amount of cartilage 3 Equal mixture of fibrous and cartilaginous tissue 4 Predominantly cartilage with small amount of fibrous tissue 5 Cartilage 6 Predominantly cartilage with small amount of immature bone 7 Equal mixture of cartilage and immature bone 8 Predominantly immature bone with small amount of cartilage 9 Union of fracture fragments by immature bone 10 10 10 10 Union of fracture fragments by mature bone defect size progress Figure Figure Figure Figure 9: percent reduction of the defect size after 12 weeks evaluated by X-ray and µCT Figure Figure Figure Figure 11 11 11 11: Histological analysis of H&E stained defect areas scored according to Huo et al. 1 Figure Figure Figure Figure 8: 12 weeks postoperative µCt showing a non-union (3 mm group) percent reduction of the defect size after 12 weeks