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Evaluation of bone regeneration using the rat critical size calvarial defect

Nature Protocols volume 7, pages 1918–1929 (2012)Cite this article

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Abstract

Animal models that are reliably reproducible, appropriate analogs to the clinical condition they are used to investigate, and that offer minimal morbidity and periprocedural mortality to the subject, are the keystone to the preclinical development of translational technologies. For bone tissue engineering, a number of small animal models exist. Here we describe the protocol for one such model, the rat calvarial defect. This versatile model allows for evaluation of biomaterials and bone tissue engineering approaches within a reproducible, non-load-bearing orthotopic site. Crucial steps for ensuring appropriate experimental control and troubleshooting tips learned through extensive experience with this model are provided. The surgical procedure itself takes ∼30 min to complete, with ∼2 h of perioperative care, and tissue collection is generally performed 4−12 weeks postoperatively. Several analytical techniques are presented, which evaluate the cellular and extracellular matrix components, functionality and mineralization, including histological, mechanical and radiographic methods.

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Figure 1: The use of the push-out jig.
Figure 2: Creation of the defect.
Figure 3: The use of the elevator.
Figure 4: Scoring guide for the extent of bony bridging and union.
Figure 5: Representative planar radiographs of defects at 12 weeks from a study looking at the release of plasmid DNA encoding BMP-2 from a hydrogel (OPF) with gelatin microparticles (CGMS).
Figure 6: Histological sections cut coronally through the defect after 12 weeks of implantation.
Figure 7: Fluorescence image of a coronally oriented histological section showing temporal fluorochrome-labeled mineral deposition.

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Acknowledgements

We acknowledge support by the US National Institutes of Health (grant nos. R21 AR56076, R01 DE015164, R01 AR42639 and R01 DE017441) for research toward the development of biomaterials for bone tissue engineering. In addition, we thank the veterinary and husbandry staff, as well as the members of the Institutional Animal Care and Use Committee at Rice University, who have had a crucial role in the development of this protocol.

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Author notes

  1. Patrick P Spicer and James D Kretlow: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Bioengineering, Rice University, Houston, Texas, USA

    Patrick P Spicer, James D Kretlow, Simon Young, F Kurtis Kasper & Antonios G Mikos

  2. Department of General Surgery, Division of Plastic Surgery, Baylor College of Medicine, Houston, Texas, USA

    James D Kretlow

  3. Department of Oral and Maxillofacial Surgery, University of Texas Health Science Center at Houston, Houston, Texas, USA

    Simon Young

  4. Department of Biomaterials, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands

    John A Jansen

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Contributions

All authors contributed equally to this work. J.D.K. and P.P.S. developed the mechanical testing methodology and wrote the manuscript. J.D.K., P.P.S., S.Y., J.A.J. and F.K.K. performed or aided in animal surgeries, microCT assessment and histological assessment. S.Y. developed the microCT and angiogenesis evaluation methodologies. J.A.J. and A.G.M. supervised all methodology development and experiments.

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Correspondence to Antonios G Mikos.

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Spicer, P., Kretlow, J., Young, S. et al. Evaluation of bone regeneration using the rat critical size calvarial defect. Nat Protoc 7, 1918–1929 (2012). https://doi.org/10.1038/nprot.2012.113

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