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

Nature Protocols volume 15, pages 877–924 (2020)Cite this article

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Abstract

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.

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Data availability

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

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Acknowledgements

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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Author notes

  1. These authors contributed equally: David S. Sparks, Siamak Saifzadeh, Flavia Medeiros Savi.

Authors and Affiliations

  1. Centre in Regenerative Medicine, Institute of Health and Biomedical Innovation, Queensland University of Technology, Kelvin Grove, Queensland, Australia

    David S. Sparks, Siamak Saifzadeh, Flavia Medeiros Savi, Constantin E. Dlaska, Arne Berner, Jan Henkel, Jiongyu Ren, Jacqui A. McGovern, Michael A. Schuetz & Dietmar W. Hutmacher

  2. Department of Plastic & Reconswrapping a sterile Coban wrap around the limb distallytructive Surgery, Princess Alexandra Hospital, Woolloongabba, Queensland, Australia

    David S. Sparks & Michael Wagels

  3. Southside Clinical Division, School of Medicine, University of Queensland, Woolloongabba, Queensland, Australia

    David S. Sparks & Michael Wagels

  4. Medical Engineering Research Facility, Queensland UCoban wrap only comes non-sterile. Sterilize Coban wrap before use.niversity of Technology, Chermside, Queensland, Australia

    Siamak Saifzadeh & Roland Steck

  5. ARC Centre for Additive Biomanufactthe mounting resin base cement. Use it only in a laboratory fume cabinet and withuring, Queensland University of Technology, Kelvin Grove, Queensland, Australia

    Flavia Medeiros Savi, Maria Ann Woodruff & Dietmar W. Hutmacher

  6. Jamieson Trauma Institute, Royal Brisbane Hospital, Herston, Queensland, Australia

    Constantin E. Dlaska, Martin Wullschleger & Michael A. Schuetz

  7. Department of Trauma Surgery, University Hospital of Regensburg, Regensburg, Germany

    Arne Berner

  8. Department of Orthopaedic Surgery, Center for Musculoskeletal Research, König-Ludwig-Haus, Julius-Maximilians-University, Würzburg, Germany

    Johannes C. Reichert

  9. Department of Orthopaedic and Trauma Surgery, Evangelisches Waldkrankenhaus Spandau, Berlin, Germany

    Johannes C. Reichert

  10. Griffith University, School of Medicine, Southport, Queensland, Australia

    Martin Wullschleger

  11. Department of Biomaterials, Max Planck Institute of Colloids and Interfaces, Potsdam, Germany

    Amaia Cipitria

  12. Australian Centre for Complex Integrated Surgical Solutions (ACCISS), Princess Alexandra Hospital, Woolloongabba, Queensland, Australia

    Michael Wagels

  13. Biofabrication and Tissue Morphology Group, Institute of Health and Biomedical Innovation, Queensland University of Technology, Kelvin Grove, Queensland, Australia

    Maria Ann Woodruff

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  1. David S. Sparks
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Contributions

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.

Ethics declarations

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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Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Related links

Key references using this protocol

Reichert, J. C. et al. Sci. Trans. Med. 4, 141ra93 (2012): https://doi.org/10.1126/scitranslmed.3003720

Berner, A. et al. Acta Biomater. 9, 7874–7884 (2013): https://doi.org/10.1016/j.actbio.2013.04.035

Berner, A. et al. J. Tissue Eng. Regen. Med. 11, 2081–2089 (2017): https://doi.org/10.1002/term.2104

Reichert, J. C. et al. Tissue Eng. B Rev. 16, 93–104 (2010): https://doi.org/10.1089/ten.TEB.2009.0455

Cipitria, A. et al. Biomaterials 34, 9960–9968 (2013): https://doi.org/10.1016/j.biomaterials.2013.09.011

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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). https://doi.org/10.1038/s41596-019-0271-2

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  • DOI: https://doi.org/10.1038/s41596-019-0271-2

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