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

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.

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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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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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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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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