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A preclinical large-animal model for the assessment of critical-size load-bearing bone defect reconstruction


Critical-size bone defects, which require large-volume tissue reconstruction, remain a clinical challenge. Bone engineering has the potential to provide new treatment concepts, yet clinical translation requires anatomically and physiologically relevant preclinical models. The ovine critical-size long-bone defect model has been validated in numerous studies as a preclinical tool for evaluating both conventional and novel bone-engineering concepts. With sufficient training and experience in large-animal studies, it is a technically feasible procedure with a high level of reproducibility when appropriate preoperative and postoperative management protocols are followed. The model can be established by following a procedure that includes the following stages: (i) preoperative planning and preparation, (ii) the surgical approach, (iii) postoperative management, and (iv) postmortem analysis. Using this model, full results for peer-reviewed publication can be attained within 2 years. In this protocol, we comprehensively describe how to establish proficiency using the preclinical model for the evaluation of a range of bone defect reconstruction options.

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Fig. 1: Schematic illustrating the process of bone defect creation and reconstruction in this large-animal model, incorporating different approaches for bone regeneration in the defect.
Fig. 2: Testing the biomechanical monitoring device fixed to the existing DCP.
Fig. 3: Instruments required for the operative procedure.
Fig. 4
Fig. 5: Positioning and preparation of the sheep for surgery.
Fig. 6: Illustration of the various key stages in the operative approach.
Fig. 7: The custom sling used for sheep limb support during the postoperative period.
Fig. 8: Mechanical testing results after 3 months.
Fig. 9: Histological and imaging analyses of bone defects after 3 and 12 months.
Fig. 10: Example specimens.
Fig. 11: Schematic overview of the histological cutting plane methodology applied to the tibial defects, paraffin and resin samples format.
Fig. 12: Morphology of newly formed bone in scaffold–rhBMP-7–treated animals.
Fig. 13: Results following micro-CT analysis.
Fig. 14: A 3-cm sheep tibia critical-size defect stained with Goldner’s trichrome, showing mineralized bone (blue) and connective tissue (orange).
Fig. 15: H&E staining showing bone morphology.
Fig. 16: Immunohistochemistry overview.
Fig. 17: Postoperative radiograph demonstrating osteosynthesis failure due to non-union in the sheep hind limb.
Fig. 18: Photographs demonstrating casting approach that lowers the risk of pressure sore development by focusing on high-risk areas.

Data availability

The datasets that support this study are available from the corresponding author upon request.


  1. 1.

    Henkel, J. et al. Bone regeneration based on tissue engineering conceptions – a 21st century perspective. Bone Res. 1, 216–248 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  2. 2.

    Weiss, R. J. et al. Decreasing incidence of tibial shaft fractures between 1998 and 2004: information based on 10,627 Swedish inpatients. Acta Orthop. 79, 526–533 (2008).

    PubMed  Google Scholar 

  3. 3.

    Wagels, M., Rowe, D., Senewiratne, S., Read, T. & Theile, D. R. Soft tissue reconstruction after compound tibial fracture: 235 cases over 12 years. J. Plast. Reconstr. Aesthet. Surg. 68, 1276–1285 (2015).

    PubMed  Google Scholar 

  4. 4.

    Wagels, M., Rowe, D., Senewiratne, S. & Theile, D. R. History of lower limb reconstruction after trauma. ANZ J. Surg. 83, 348–353 (2013).

    PubMed  Google Scholar 

  5. 5.

    Sparks, D. S. et al. Vascularised bone transfer: history, blood supply and contemporary problems. J. Plast. Reconstr. Aesthet. Surg. 70, 1–11 (2017).

    PubMed  Google Scholar 

  6. 6.

    Reichert, J. C. et al. A tissue engineering solution for segmental defect regeneration in load-bearing long bones. Sci. Transl. Med. 4, 141ra193 (2012).

    Google Scholar 

  7. 7.

    Schmitz, J. P. & Hollinger, J. O. The critical size defect as an experimental model for craniomandibulofacial nonunions. Clin. Orthop. Relat. Res. 1986, 299-308 (1986).

  8. 8.

    Berner, A. et al. Treatment of long bone defects and non-unions: from research to clinical practice. Cell Tissue Res. 347, 501–519 (2012).

    PubMed  Google Scholar 

  9. 9.

    Reichert, J. C. et al. The challenge of establishing preclinical models for segmental bone defect research. Biomaterials 30, 2149–2163 (2009).

    CAS  PubMed  Google Scholar 

  10. 10.

    Balogh, Z. J. et al. Advances and future directions for management of trauma patients with musculoskeletal injuries. Lancet 380, 1109–1119 (2012).

    PubMed  Google Scholar 

  11. 11.

    Sparks, D. S., Wagels, M. & Taylor, G. I. Bone reconstruction: a history of vascularized bone transfer. Microsurgery 38, 7–13 (2018).

    PubMed  Google Scholar 

  12. 12.

    Cipitria, A. et al. Polycaprolactone scaffold and reduced rhBMP-7 dose for the regeneration of critical-sized defects in sheep tibiae. Biomaterials 34, 9960–9968 (2013).

    CAS  PubMed  Google Scholar 

  13. 13.

    Berner, A. et al. Biomimetic tubular nanofiber mesh and platelet rich plasma-mediated delivery of BMP-7 for large bone defect regeneration. Cell Tissue Res. 347, 603–612 (2012).

    CAS  PubMed  Google Scholar 

  14. 14.

    Reichert, J. C. et al. Custom-made composite scaffolds for segmental defect repair in long bones. Int. Orthop. 35, 1229–1236 (2011).

    PubMed  Google Scholar 

  15. 15.

    Berner, A. et al. Effects of scaffold architecture on cranial bone healing. Int. J. Oral. Maxillofac. Surg. 43, 506–513 (2014).

    CAS  PubMed  Google Scholar 

  16. 16.

    Breschi, A., Gingeras, T. R. & Guigo, R. Comparative transcriptomics in human and mouse. Nat. Rev. Genet. 18, 425–440 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  17. 17.

    Wancket, L. M. Animal models for evaluation of bone implants and devices: comparative bone structure and common model uses. Vet. Pathol. 52, 842–850 (2015).

    CAS  PubMed  Google Scholar 

  18. 18.

    Aerssens, J., Boonen, S., Lowet, G. & Dequeker, J. Interspecies differences in bone composition, density, and quality: potential implications for in vivo bone research. Endocrinology 139, 663–670 (1998).

    CAS  PubMed  Google Scholar 

  19. 19.

    Wang, X., Mabrey, J. D. & Agrawal, C. M. An interspecies comparison of bone fracture properties. Biomed. Mater. Eng. 8, 1–9 (1998).

    CAS  PubMed  Google Scholar 

  20. 20.

    Reichert, J. C. et al. Establishment of a preclinical ovine model for tibial segmental bone defect repair by applying bone tissue engineering strategies. Tissue Eng. Part B Rev. 16, 93–104 (2010).

    PubMed  Google Scholar 

  21. 21.

    McGovern, J. A., Griffin, M. & Hutmacher, D. W. Animal models for bone tissue engineering and modelling disease. Dis. Model. Mech. 11, 33084 (2018).

  22. 22.

    Pearce, A. I., Richards, R. G., Milz, S., Schneider, E. & Pearce, S. G. Animal models for implant biomaterial research in bone: a review. Eur. Cell. Mater. 13, 1–10 (2007).

    CAS  PubMed  Google Scholar 

  23. 23.

    Martini, L., Fini, M., Giavaresi, G. & Giardino, R. Sheep model in orthopedic research: a literature review. Comp. Med. 51, 292–299 (2001).

    CAS  PubMed  Google Scholar 

  24. 24.

    Hu, Y., Zhang, C., Zhang, S., Xiong, Z. & Xu, J. Development of a porous poly(L-lactic acid)/hydroxyapatite/collagen scaffold as a BMP delivery system and its use in healing canine segmental bone defect. J. Biomed. Mater. Res. A 67, 591–598 (2003).

    PubMed  Google Scholar 

  25. 25.

    Theyse, L. F., Oosterlaken-Dijksterhuis, M. A., van Doorn, J., Dhert, W. J. & Hazewinkel, H. A. Growth hormone stimulates bone healing in a critical-sized bone defect model. Clin. Orthop. Relat. Res. 446, 259–267 (2006).

    CAS  PubMed  Google Scholar 

  26. 26.

    Tiedeman, J. J., Lippiello, L., Connolly, J. F. & Strates, B. S. Quantitative roentgenographic densitometry for assessing fracture healing. Clin. Orthop. Relat. Res. 1990, 279-286 (1990).

  27. 27.

    Takigami, H. et al. Bone formation following OP-1 implantation is improved by addition of autogenous bone marrow cells in a canine femur defect model. J. Orthop. Res. 25, 1333–1342 (2007).

    PubMed  Google Scholar 

  28. 28.

    Welter, J. F. et al. Cyclosporin A and tissue antigen matching in bone transplantation. Fibular allografts studied in the dog. Acta Orthop. Scand. 61, 517–527 (1990).

    CAS  PubMed  Google Scholar 

  29. 29.

    Pek, Y. S., Gao, S., Arshad, M. S., Leck, K. J. & Ying, J. Y. Porous collagen-apatite nanocomposite foams as bone regeneration scaffolds. Biomaterials 29, 4300–4305 (2008).

    CAS  PubMed  Google Scholar 

  30. 30.

    Meinig, R. P., Buesing, C. M., Helm, J. & Gogolewski, S. Regeneration of diaphyseal bone defects using resorbable poly(L/DL-lactide) and poly(D-lactide) membranes in the Yucatan pig model. J. Orthop. Trauma 11, 551–558 (1997).

    CAS  PubMed  Google Scholar 

  31. 31.

    Anderson, M. L. et al. Critical size defect in the goat’s os ilium. A model to evaluate bone grafts and substitutes. Clin. Orthop. Relat. Res. 1999, 231-239 (1999).

  32. 32.

    Leung, K. S. et al. Goats as an osteopenic animal model. J. Bone Miner. Res. 16, 2348–2355 (2001).

    CAS  PubMed  Google Scholar 

  33. 33.

    Wang, L. et al. Osteogenesis and angiogenesis of tissue-engineered bone constructed by prevascularized β-tricalcium phosphate scaffold and mesenchymal stem cells. Biomaterials 31, 9452–9461 (2010).

    CAS  PubMed  Google Scholar 

  34. 34.

    Liu, G. et al. Repair of goat tibial defects with bone marrow stromal cells and beta-tricalcium phosphate. J. Mater. Sci. Mater. Med. 19, 2367–2376 (2008).

    CAS  PubMed  Google Scholar 

  35. 35.

    Nandi, S. K., Kundu, B., Datta, S., De, D. K. & Basu, D. The repair of segmental bone defects with porous bioglass: an experimental study in goat. Res. Vet. Sci. 86, 162–173 (2009).

    CAS  PubMed  Google Scholar 

  36. 36.

    Qin, L., Mak, A. T., Cheng, C. W., Hung, L. K. & Chan, K. M. Histomorphological study on pattern of fluid movement in cortical bone in goats. Anat. Rec. 255, 380–387 (1999).

    CAS  PubMed  Google Scholar 

  37. 37.

    Newman, E., Turner, A. S. & Wark, J. D. The potential of sheep for the study of osteopenia: current status and comparison with other animal models. Bone 16, 277S–284S (1995).

    CAS  PubMed  Google Scholar 

  38. 38.

    Taylor, W. R. et al. Tibio-femoral joint contact forces in sheep. J. Biomech. 39, 791–798 (2006).

    PubMed  Google Scholar 

  39. 39.

    Ravaglioli, A. et al. Mineral evolution of bone. Biomaterials 17, 617–622 (1996).

    CAS  PubMed  Google Scholar 

  40. 40.

    Lutton, C. et al. Transplanted abdominal granulation tissue induced bone formation—an in vivo study in sheep. Connect. Tissue Res. 50, 256–262 (2009).

    CAS  PubMed  Google Scholar 

  41. 41.

    Jones, C. W. et al. Matrix-induced autologous chondrocyte implantation in sheep: objective assessments including confocal arthroscopy. J. Orthop. Res. 26, 292–303 (2008).

    CAS  PubMed  Google Scholar 

  42. 42.

    Nuss, K. M., Auer, J. A., Boos, A. & von Rechenberg, B. An animal model in sheep for biocompatibility testing of biomaterials in cancellous bones. BMC Musculoskelet. Disord. 7, 67 (2006).

    PubMed  PubMed Central  Google Scholar 

  43. 43.

    Wieding, J., Lindner, T., Bergschmidt, P. & Bader, R. Biomechanical stability of novel mechanically adapted open-porous titanium scaffolds in metatarsal bone defects of sheep. Biomaterials 46, 35–47 (2015).

    CAS  PubMed  Google Scholar 

  44. 44.

    Viateau, V. et al. Induction of a barrier membrane to facilitate reconstruction of massive segmental diaphyseal bone defects: an ovine model. Vet. Surg. 35, 445–452 (2006).

    PubMed  Google Scholar 

  45. 45.

    Li, Z. et al. Repair of sheep metatarsus defects by using tissue-engineering technique. J. Huazhong Univ. Sci. Technol. Med. Sci. 25, 62–67 (2005).

    PubMed  Google Scholar 

  46. 46.

    Filardo, G. et al. Vegetable hierarchical structures as template for bone regeneration: new bio-ceramization process for the development of a bone scaffold applied to an experimental sheep model. J. Biomed. Mater. Res. B Appl. Biomater. (2019).

  47. 47.

    Li, J. J. et al. A novel bone substitute with high bioactivity, strength, and porosity for repairing large and load-bearing bone defects. Adv. Healthc. Mater. 8, e1900641 (2019).

    PubMed  Google Scholar 

  48. 48.

    Kirby, G. T. S. et al. Microparticles for sustained growth factor delivery in the regeneration of critically-sized segmental tibial bone defects. Materials (Basel) 9, 259 (2016).

    PubMed Central  Google Scholar 

  49. 49.

    Li, J. J. et al. Efficacy of novel synthetic bone substitutes in the reconstruction of large segmental bone defects in sheep tibiae. Biomed. Mater. 11, 015016 (2016).

    PubMed  Google Scholar 

  50. 50.

    Berner, A. et al. Scaffold-cell bone engineering in a validated preclinical animal model: precursors vs differentiated cell source. J. Tissue Eng. Regen. Med. 11, 2081–2089 (2017).

    CAS  PubMed  Google Scholar 

  51. 51.

    Berner, A. et al. Delayed minimally invasive injection of allogenic bone marrow stromal cell sheets regenerates large bone defects in an ovine preclinical animal model. Stem Cells Transl. Med. 4, 503–512 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  52. 52.

    Berner, A. et al. Autologous vs. allogenic mesenchymal progenitor cells for the reconstruction of critical sized segmental tibial bone defects in aged sheep. Acta Biomater. 9, 7874–7884 (2013).

    CAS  PubMed  Google Scholar 

  53. 53.

    Cipitria, A. et al. BMP delivery complements the guiding effect of scaffold architecture without altering bone microstructure in critical-sized long bone defects: a multiscale analysis. Acta Biomater. 23, 282–294 (2015).

    CAS  PubMed  Google Scholar 

  54. 54.

    Croker, S. L., Reed, W. & Donlon, D. Comparative cortical bone thickness between the long bones of humans and five common non-human mammal taxa. Forensic Sci. Int. 260, 104 e101–104 e117 (2016).

    Google Scholar 

  55. 55.

    Singh, B. Dyce, Sack and Wensing’s Textbook on Veterinary Anatomy (Elsevier, 2017).

  56. 56.

    Connolly, J. F., Guse, R., Tiedeman, J. & Dehne, R. Autologous marrow injection as a substitute for operative grafting of tibial nonunions. Clin. Orthop. Relat. Res. 1991, 259-270 (1991).

  57. 57.

    Xerogeanes, J. W. et al. A functional comparison of animal anterior cruciate ligament models to the human anterior cruciate ligament. Ann. Biomed. Eng. 26, 345–352 (1998).

    CAS  PubMed  Google Scholar 

  58. 58.

    Seebeck, P. et al. Gait evaluation: a tool to monitor bone healing? Clin. Biomech. (Bristol, Avon) 20, 883–891 (2005).

    CAS  Google Scholar 

  59. 59.

    Ebraheim, N. A. et al. Comparison of intramedullary nail, plate, and external fixation in the treatment of distal tibia nonunions. Int. Orthop. 41, 1925–1934 (2017).

    PubMed  Google Scholar 

  60. 60.

    Tufekci, P. et al. Early mechanical stimulation only permits timely bone healing in sheep. J. Orthop. Res. 36, 1790–1796 (2018).

    PubMed  Google Scholar 

  61. 61.

    Pobloth, A. M. et al. Tubular open-porous β-tricalcium phosphate polycaprolactone scaffolds as guiding structure for segmental bone defect regeneration in a novel sheep model. J. Tissue Eng. Regen. Med. 12, 897–911 (2018).

    CAS  PubMed  Google Scholar 

  62. 62.

    Fernandes, M. B. et al. The effect of bone allografts combined with bone marrow stromal cells on the healing of segmental bone defects in a sheep model. BMC Vet. Res. 10, 36 (2014).

    PubMed  PubMed Central  Google Scholar 

  63. 63.

    Gugala, Z. & Gogolewski, S. Healing of critical-size segmental bone defects in the sheep tibiae using bioresorbable polylactide membranes. Injury 33 (Suppl. 2), B71–B76 (2002).

    PubMed  Google Scholar 

  64. 64.

    Maissen, O. et al. Mechanical and radiological assessment of the influence of rhTGFbeta-3 on bone regeneration in a segmental defect in the ovine tibia: pilot study. J. Orthop. Res. 24, 1670–1678 (2006).

    CAS  PubMed  Google Scholar 

  65. 65.

    Herten, M. et al. Biomechanical stability and osteogenesis in a tibial bone defect treated by autologous ovine cord blood cells—a pilot study. Molecules 24, 295 (2019).

    PubMed Central  Google Scholar 

  66. 66.

    Seebeck, P. et al. Do serological tissue turnover markers represent callus formation during fracture healing? Bone 37, 669–677 (2005).

    CAS  PubMed  Google Scholar 

  67. 67.

    Haubruck, P. et al. Complications and risk management in the use of the reaming-irrigator-aspirator (RIA) system: RIA is a safe and reliable method in harvesting autologous bone graft. PLoS One 13, e0196051 (2018).

    PubMed  PubMed Central  Google Scholar 

  68. 68.

    Ochsner, P. E., Baumgart, F. & Kohler, G. Heat-induced segmental necrosis after reaming of one humeral and two tibial fractures with a narrow medullary canal. Injury 29(Suppl. 2), B1–B10 (1998).

    PubMed  Google Scholar 

  69. 69.

    Christou, C., Oliver, R. A., Yu, Y. & Walsh, W. R. The Masquelet technique for membrane induction and the healing of ovine critical sized segmental defects. PLoS One 9, e114122 (2014).

    PubMed  PubMed Central  Google Scholar 

  70. 70.

    Sarkar, M. R. et al. Bone formation in a long bone defect model using a platelet-rich plasma-loaded collagen scaffold. Biomaterials 27, 1817–1823 (2006).

    CAS  PubMed  Google Scholar 

  71. 71.

    Tyllianakis, M. et al. Biomechanical comparison of callus over a locked intramedullary nail in various segmental bone defects in a sheep model. Med. Sci. Monit. 13, BR125–BR130 (2007).

    PubMed  Google Scholar 

  72. 72.

    Schneiders, W. et al. In vivo effects of modification of hydroxyapatite/collagen composites with and without chondroitin sulphate on bone remodeling in the sheep tibia. J. Orthop. Res. 27, 15–21 (2009).

    CAS  PubMed  Google Scholar 

  73. 73.

    Bloemers, F. W. et al. Autologous bone versus calcium-phosphate ceramics in treatment of experimental bone defects. J. Biomed. Mater. Res. B Appl. Biomater. 66, 526–531 (2003).

    PubMed  Google Scholar 

  74. 74.

    Hahn, J. A., Witte, T. S., Arens, D., Pearce, A. & Pearce, S. Double-plating of ovine critical sized defects of the tibia: a low morbidity model enabling continuous in vivo monitoring of bone healing. BMC Musculoskelet. Disord. 12, 214 (2011).

    PubMed  PubMed Central  Google Scholar 

  75. 75.

    Niemeyer, P. et al. Xenogenic transplantation of human mesenchymal stem cells in a critical size defect of the sheep tibia for bone regeneration. Tissue Eng. Part A 16, 33–43 (2010).

    CAS  PubMed  Google Scholar 

  76. 76.

    Mastrogiacomo, M. et al. Reconstruction of extensive long bone defects in sheep using resorbable bioceramics based on silicon stabilized tricalcium phosphate. Tissue Eng. 12, 1261–1273 (2006).

    CAS  PubMed  Google Scholar 

  77. 77.

    Niemeyer, P. et al. Comparison of mesenchymal stem cells from bone marrow and adipose tissue for bone regeneration in a critical size defect of the sheep tibia and the influence of platelet-rich plasma. Biomaterials 31, 3572–3579 (2010).

    CAS  PubMed  Google Scholar 

  78. 78.

    Pobloth, A. M. et al. Mechanobiologically optimized 3D titanium-mesh scaffolds enhance bone regeneration in critical segmental defects in sheep. Sci. Transl. Med. 10, aam8828 (2018).

    PubMed  Google Scholar 

  79. 79.

    Giannoni, P. et al. Regeneration of large bone defects in sheep using bone marrow stromal cells. J. Tissue Eng. Regen. Med. 2, 253–262 (2008).

    CAS  PubMed  Google Scholar 

  80. 80.

    Chehade, M. J., Pohl, A. P., Pearcy, M. J. & Nawana, N. Clinical implications of stiffness and strength changes in fracture healing. J. Bone Jt. Surg. Br. 79, 9–12 (1997).

    CAS  Google Scholar 

  81. 81.

    Augat, P. et al. Shear movement at the fracture site delays healing in a diaphyseal fracture model. J. Orthop. Res. 21, 1011–1017 (2003).

    PubMed  Google Scholar 

  82. 82.

    Claes, L. et al. Early dynamization by reduced fixation stiffness does not improve fracture healing in a rat femoral osteotomy model. J. Orthop. Res. 27, 22–27 (2009).

    PubMed  Google Scholar 

  83. 83.

    Steiner, M. et al. Comparison between different methods for biomechanical assessment of ex vivo fracture callus stiffness in small animal bone healing studies. PLoS One 10, e0119603 (2015).

    PubMed  PubMed Central  Google Scholar 

  84. 84.

    Drakonaki, E. E., Allen, G. M. & Wilson, D. J. Ultrasound elastography for musculoskeletal applications. Br. J. Radiol. 85, 1435–1445 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  85. 85.

    Ekeland, A., Engesaeter, L. B. & Langeland, N. Mechanical properties of fractured and intact rat femora evaluated by bending, torsional and tensile tests. Acta Orthop. Scand. 52, 605–613 (1981).

    CAS  PubMed  Google Scholar 

  86. 86.

    Seide, K. et al. Telemetric assessment of bone healing with an instrumented internal fixator: a preliminary study. J. Bone Jt. Surg. Br. 94, 398–404 (2012).

    CAS  Google Scholar 

  87. 87.

    Burny, F. et al. Concept, design and fabrication of smart orthopedic implants. Med. Eng. Phys. 22, 469–479 (2000).

    CAS  PubMed  Google Scholar 

  88. 88.

    Fountain, S. et al. Monitoring healing progression and characterizing the mechanical environment in preclinical models for bone tissue engineering. Tissue Eng. Part B Rev. 22, 47–57 (2016).

    PubMed  Google Scholar 

  89. 89.

    ASTM. Standard Guide for Pre-clinical in vivo Evaluation in Critical Size Segmental Bone Defects. (ASTM International, 2014).

  90. 90.

    Christou, C., Oliver, R. A., Rawlinson, J. & Walsh, W. R. Transdermal fentanyl and its use in ovine surgery. Res. Vet. Sci. 100, 252–256 (2015).

    CAS  PubMed  Google Scholar 

  91. 91.

    Cocquyt, G., Driessen, B. & Simoens, P. Variability in the eruption of the permanent incisor teeth in sheep. Vet. Rec. 157, 619–623 (2005).

    CAS  PubMed  Google Scholar 

  92. 92.

    Berry, S. H. Injectable anesthetics in Veterinary Anesthesia and Analgesia: The Fifth Edition of Lumb and Jones (eds K. A. Grimm et al.) (Wiley, 2015).

  93. 93.

    Goldberg, M. E. & Shaffran, N. Pain Management for Veterinary Technicians and Nurses (Wiley-Blackwell, 2014).

  94. 94.

    Quiros-Carmona, S. et al. A comparison of cardiopulmonary effects and anaesthetic requirements of two dexmedetomidine continuous rate infusions in alfaxalone-anaesthetized greyhounds. Vet. Anaesth. Analg. 44, 228–236 (2017).

    CAS  PubMed  Google Scholar 

  95. 95.

    Funes, F. J. et al. Anaesthetic and cardiorespiratory effects of a constant rate infusion of fentanyl in isoflurane-anaesthetized sheep. Vet. Anaesth. Analg. 42, 157–164 (2015).

    CAS  PubMed  Google Scholar 

  96. 96.

    Welsh, E. M., McKellar, Q. A. & Nolan, A. M. The pharmacokinetics of flunixin meglumine in the sheep. J. Vet. Pharmacol. Ther. 16, 181–188 (1993).

    CAS  PubMed  Google Scholar 

  97. 97.

    Blackburn, P. J., Carmichael, I. H., Walkden-Brown, S. W. & Greenslade, S. Cost of immune response to Trichostrongylus vitrinus infection in meat sheep. in Proceedings of the Australian Sheep Veterinarians, 2013 Conference (Australian Sheep Veterinarians, 2014).

  98. 98.

    Elsheikh, H. A., Osman, I. A. & Ali, B. H. Comparative pharmacokinetics of ampicillin trihydrate, gentamicin sulphate and oxytetracycline hydrochloride in Nubian goats and desert sheep. J. Vet. Pharmacol. Ther. 20, 262–266 (1997).

    CAS  PubMed  Google Scholar 

  99. 99.

    Reilly, J. S. Euthanasia of Animals Used for Scientific Purposes 2nd edn (Australian and New Zealand Council for the Care of Animals in Research and Teaching (ANZCCART), 2001).

  100. 100.

    Oetzel, G. R. Parturient paresis and hypocalcemia in ruminant livestock. Vet. Clin. North Am. Food Anim. Pract. 4, 351–364 (1988).

    CAS  PubMed  Google Scholar 

  101. 101.

    Nair, J. et al. Bioavailability of endotracheal epinephrine in an ovine model of neonatal resuscitation. Early Hum. Dev. 130, 27–32 (2019).

    CAS  PubMed  PubMed Central  Google Scholar 

  102. 102.

    Lorenz, I. & Lorch, A. D-lactic acidosis in lambs. Vet. Rec. 164, 174–175 (2009).

    CAS  PubMed  Google Scholar 

  103. 103.

    Lankadeva, Y. R. et al. Strategies that improve renal medullary oxygenation during experimental cardiopulmonary bypass may mitigate postoperative acute kidney injury. Kidney Int. 95, 1338–1346 (2019).

    PubMed  Google Scholar 

  104. 104.

    Passmore, M. R. et al. Fluid resuscitation with 0.9% saline alters haemostasis in an ovine model of endotoxemic shock. Thromb. Res. 176, 39–45 (2019).

    CAS  PubMed  Google Scholar 

  105. 105.

    Dardenne, A. et al. Benefits of standardizing the treatment of arrhythmias in the sheep (Ovis aries) model of chronic heart failure after myocardial infarction. J. Am. Assoc. Lab. Anim. Sci. 52, 290–294 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  106. 106.

    Schauvliege, S. et al. Refined anaesthesia for implantation of engineered experimental aortic valves in the pulmonary artery using a right heart bypass in sheep. Lab. Anim. 40, 341–352 (2006).

    CAS  PubMed  Google Scholar 

  107. 107.

    Letzelter, J., Hill, J. B. & Hacquebord, J. An overview of skin antiseptics used in orthopaedic surgery procedures. J. Am. Acad. Orthop. Surg. 27, 599–606 (2019).

    PubMed  Google Scholar 

  108. 108.

    Zhou, H., Ge, J., Bai, Y., Liang, C. & Yang, L. Translation of bone wax and its substitutes: history, clinical status and future directions. J. Orthop. Transl. 17, 64–72 (2019).

    Google Scholar 

  109. 109.

    Lacasta, D. et al. Vaccination schedules in small ruminant farms. Vet. Microbiol. 181, 34–46 (2015).

    CAS  PubMed  Google Scholar 

  110. 110.

    Leathwick, D. M. et al. Drenching adult ewes: implications of anthelmintic treatments pre- and post-lambing on the development of anthelmintic resistance. NZ Vet. J. 54, 297–304 (2006).

    CAS  Google Scholar 

  111. 111.

    Araco, A., Caruso, R., Araco, F., Overton, J. & Gravante, G. Capsular contractures: a systematic review. Plast. Reconstr. Surg. 124, 1808–1819 (2009).

    CAS  PubMed  Google Scholar 

  112. 112.

    Pass, M. A. & Mogg, T. D. Pharmacokinetics and metabolism of amitraz in ponies and sheep. J. Vet. Pharmacol. Ther. 18, 210–215 (1995).

    CAS  PubMed  Google Scholar 

  113. 113.

    Grimm, K. A., Lamont, L. A., Tranquilli, W. J., Greene, S. A. & Robertson, S. A. Veterinary Anesthesia and Analgesia: The Fifth Edition of Lumb and Jones (Wiley, 2015).

  114. 114.

    Seldinger, A. I. Catheter replacement of the needle in percutaneous arteriography: a new technique. Acta Radiol. 39, 368–376 (1953).

    CAS  PubMed  Google Scholar 

  115. 115.

    Weingart, S. D. & Levitan, R. M. Preoxygenation and prevention of desaturation during emergency airway management. Ann. Emerg. Med. 59, 165–175 e161 (2012).

    PubMed  Google Scholar 

  116. 116.

    Willbold, E. & Witte, F. Histology and research at the hard tissue-implant interface using Technovit 9100 new embedding technique. Acta Biomater. 6, 4447–4455 (2010).

    CAS  PubMed  Google Scholar 

  117. 117.

    Donath, K. Preparation of histologic sections by a cutting-grinding technique for hard tissue and other materials not suitable to be sectioned by routine methods. in Equipment and Methodological Performance 2nd edn (EXAKT-Kulzer, 1995).

  118. 118.

    Savi, F. M., Brierly, G. I., Baldwin, J., Theodoropoulos, C. & Woodruff, M. A. Comparison of different decalcification methods using rat mandibles as a model. J. Histochem. Cytochem. 65, 705–722 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  119. 119.

    Li, J. J. et al. Effects of material-tissue interactions on bone regeneration outcomes using baghdadite implants in a large animal model. Adv. Healthc. Mater. 7, e1800218 (2018).

    PubMed  Google Scholar 

  120. 120.

    Paris, M. et al. Scaffold curvature-mediated novel biomineralization process originates a continuous soft tissue-to-bone interface. Acta Biomater. 60, 64–80 (2017).

    PubMed  Google Scholar 

  121. 121.

    Muschler, G. F., Raut, V. P., Patterson, T. E., Wenke, J. C. & Hollinger, J. O. The design and use of animal models for translational research in bone tissue engineering and regenerative medicine. Tissue Eng. Part B Rev. 16, 123–145 (2010).

    PubMed  Google Scholar 

  122. 122.

    Li, Y. et al. Bone defect animal models for testing efficacy of bone substitute biomaterials. J. Orthop. Transl. 3, 95–104 (2015).

    Google Scholar 

  123. 123.

    Mosekilde, L., Kragstrup, J. & Richards, A. Compressive strength, ash weight, and volume of vertebral trabecular bone in experimental fluorosis in pigs. Calcif. Tissue Int. 40, 318–322 (1987).

    CAS  PubMed  Google Scholar 

  124. 124.

    Dias, I. R. et al. Preclinical and translational studies in small ruminants (sheep and goat) as models for osteoporosis research. Curr. Osteoporos. Rep. 16, 182–197 (2018).

    PubMed  Google Scholar 

  125. 125.

    Moran, C. J. et al. The benefits and limitations of animal models for translational research in cartilage repair. J. Exp. Orthop. 3, 1 (2016).

    PubMed  PubMed Central  Google Scholar 

  126. 126.

    Fountain, S. M. Monitoring Healing Progression and Characterising the Mechanical Environment in a Preclinical Bone Defect Model. PhD thesis, Queensland University of Technology (2016).

  127. 127.

    Yong, M. R. et al. Establishment and characterization of an open mini-thoracotomy surgical approach to an ovine thoracic spine fusion model. Tissue Eng. Part C. Methods 20, 19–27 (2014).

    CAS  PubMed  Google Scholar 

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This work was supported by the German Research Foundation (DFG; BE 4492/1-2 and HE 7074/1-1) and the Australian Research Council (ARC LP100200084, ARC IC160100026, Industrial Transformation Training Centre in Additive Biomanufacturing, ARC Future Fellowship awarded to D.W.H.). This work was also supported by funding through the Wesley Hospital Foundation, the AO Foundation and the Princess Alexandra Hospital Research Foundation. We thank the staff at the Queensland University of Technology (QUT) Medical Engineering Research Facility for veterinary assistance and administrative and technical support.

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D.S.S. wrote the manuscript with the assistance of S.S., F.M.S., J.R., J.A.M. and D.W.H. S.S. and J.C.R. designed and performed the experiments, and S.S. provided veterinary expertise. D.S.S., F.M.S., A.C., C.E.D., A.B., J.H., J.C.R., M. Wullschleger and J.R. performed the experiments and prepared and analyzed the data. R.S. designed, performed and analyzed the biomechanical testing. S.S., M. Wagels, M.A.W., M.A.S. and D.W.H. supervised the project, M.A.S. and M. Wagels provided clinical input into the design of the model. D.W.H. led the design of the experiments and supervised the project. All authors read and critiqued the manuscript extensively and agreed on the final version of the manuscript

Corresponding author

Correspondence to Dietmar W. Hutmacher.

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Competing interests

D.W.H. is a cofounder and shareholder of Osteopore International Pty Ltd, a company specializing in 3D bioresorbable implants to assist with bone healing.

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Key references using this protocol

Reichert, J. C. et al. Sci. Trans. Med. 4, 141ra93 (2012):

Berner, A. et al. Acta Biomater. 9, 7874–7884 (2013):

Berner, A. et al. J. Tissue Eng. Regen. Med. 11, 2081–2089 (2017):

Reichert, J. C. et al. Tissue Eng. B Rev. 16, 93–104 (2010):

Cipitria, A. et al. Biomaterials 34, 9960–9968 (2013):

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Sparks, D.S., Saifzadeh, S., Savi, F.M. et al. A preclinical large-animal model for the assessment of critical-size load-bearing bone defect reconstruction. Nat Protoc 15, 877–924 (2020).

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