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A composite critical-size rabbit mandibular defect for evaluation of craniofacial tissue regeneration

Nature Protocols volume 11, pages 1989–2009 (2016)Cite this article

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Abstract

Translational biomaterials targeted toward the regeneration of large bone defects in the mandible require a preclinical model that accurately recapitulates the regenerative challenges present in humans. Computational modeling and in vitro assays do not fully replicate the in vivo environment. Consequently, in vivo models can have specific applications such as those of the mandibular angle defect, which is used to investigate bone regeneration in a nonload-bearing area, and the inferior border mandibular defect, which is a model for composite bone and nerve regeneration, with both models avoiding involvement of soft tissue or teeth. In this protocol, we describe a reproducible load-bearing critical-size composite tissue defect comprising loss of soft tissue, bone and tooth in the mandible of a rabbit. We have previously used this procedure to investigate bone regeneration, vascularization and infection prevention in response to new biomaterial formulations for craniofacial tissue engineering applications. This surgical approach can be adapted to investigate models such as that of regeneration in the context of osteoporosis or irradiation. The procedure can be performed by researchers with basic surgical skills such as dissection and suturing. The procedure takes 1.5–2 h, with ∼2 h of immediate postoperative care, and animals should be monitored daily for the remainder of the study. For bone tissue engineering applications, tissue collection typically occurs 12 weeks after surgery. In this protocol, we will present the necessary steps to ensure reproducibility; tips to minimize complications during and after surgery; and analytical techniques for assessing soft tissue, bone and vessel regeneration by gross evaluation, microcomputed tomography (microCT) and histology.

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Figure 1: Overview of the rabbit mandibular defect procedure.
Figure 2: Flowchart for sample preparation.
Figure 3: Dissection of the mandible.
Figure 4: Creation of the mandibular defect.
Figure 5: Placement of fixation hardware.
Figure 6: Creation of the intraoral communication.
Figure 7: Microfil infusion.
Figure 8: Gross evaluation of soft-tissue healing at 12 weeks after surgery.
Figure 9: Representative microCT images of vasculature surrounding an implant in a full-thickness defect without intraoral communication.
Figure 10: Representative microCT scoring images (as described in Supplementary Table 1) of bone regeneration over a polymethylmethacrylate implant.
Figure 11: Representative histologic images and scoring histograms (as described in Supplementary Table 2).

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References

  1. Smith, B.T., Shum, J., Wong, M., Mikos, A.G. & Young, S. Bone tissue engineering challenges in oral & maxillofacial surgery. Adv. Exp. Med. Biol. 881, 57–78 (2015).

    CAS  PubMed  Google Scholar 

  2. Schmitz, J.P. & Hollinger, J.O. The critical size defect as an experimental-model for craniomandibulofacial nonunions. Clin. Orthop. Relat. Res. 205, 299–308 (1986).

    Google Scholar 

  3. Takagi, K. & Urist, M.R. The reaction of the dura to bone morphogenetic protein (Bmp) in repair of skull defects. Ann. Surg. 196, 100–109 (1982).

    CAS  PubMed  PubMed Central  Google Scholar 

  4. Young, S. et al. Development and characterization of a rabbit alveolar bone nonhealing defect model. J. Biomed. Mater. Res. A 86A, 182–194 (2008).

    CAS  Google Scholar 

  5. Spicer, P.P. et al. Evaluation of bone regeneration using the rat critical size calvarial defect. Nat. Protoc. 7, 1918–1929 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  6. Cooper, G.M. et al. Testing the critical size in calvarial bone defects: revisiting the concept of a critical-size defect. Plast. Reconstr. Surg. 125, 1685–1692 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  7. Kasper, F.K., Tanahashi, K., Fisher, J.P. & Mikos, A.G. Synthesis of poly(propylene fumarate). Nat. Protoc. 4, 518–525 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  8. Kinard, L.A., Kasper, F.K. & Mikos, A.G. Synthesis of oligo(poly(ethylene glycol) fumarate). Nat. Protoc. 7, 1219–1227 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  9. Watson, B.M. et al. Biodegradable, phosphate-containing, dual-gelling macromers for cellular delivery in bone tissue engineering. Biomaterials 67, 286–296 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  10. Sanz-Herrera, J.A. & Reina-Romo, E. Cell-biomaterial mechanical interaction in the framework of tissue engineering: insights, computational modeling and perspectives. Int. J. Mol. Sci. 12, 8217–8244 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  11. Bancroft, G.N., Sikavitsas, V.I. & Mikos, A.G. Design of a flow perfusion bioreactor system for bone tissue-engineering applications. Tissue Eng. 9, 549–554 (2003).

    CAS  PubMed  Google Scholar 

  12. Tatara, A.M., Shah, S.R., Livingston, C.E. & Mikos, A.G. Infected animal models for tissue engineering. Methods 84, 17–24 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  13. Mankani, M.H., Kuznetsov, S.A., Wolfe, R.M., Marshall, G.W. & Robey, P.G. In vivo bone formation by human bone marrow stromal cells: reconstruction of the mouse calvarium and mandible. Stem Cells 24, 2140–2149 (2006).

    PubMed  Google Scholar 

  14. Saadeh, P.B. et al. Repair of a critical size defect in the rat mandible using allogenic type I collagen. J. Craniofac. Surg. 12, 573–579 (2001).

    CAS  PubMed  Google Scholar 

  15. van Leeuwen, A.C. et al. Guided bone regeneration in rat mandibular defects using resorbable poly(trimethylene carbonate) barrier membranes. Acta Biomater. 8, 1422–1429 (2012).

    CAS  PubMed  Google Scholar 

  16. Stubinger, S. & Dard, M. The rabbit as experimental model for research in implant dentistry and related tissue regeneration. J. Invest. Surg. 26, 266–282 (2013).

    PubMed  Google Scholar 

  17. Elsalanty, M.E. et al. Reconstruction of canine mandibular bone defects using a bone transport reconstruction plate. Ann. Plast. Surg. 63, 441–448 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  18. Nienhuijs, M.E.L. et al. Healing of bone defects in the goat mandible, using COLLOSS (R) E and beta-tricalciumphosphate. J. Biomed. Mater. Res. B 92B, 517–524 (2010).

    CAS  Google Scholar 

  19. Zhou, L.B. et al. Reconstruction of curved mandibular angle defects using a new internal transport distraction device: an experiment in goats. Br. J. Oral Maxillofac. Surg. 46, 445–448 (2008).

    PubMed  Google Scholar 

  20. Ma, J.L., Pan, J.L., Tan, B.S. & Cui, F.Z. Determination of critical size defect of minipig mandible. J. Tissue Eng. Regen. Med. 3, 615–622 (2009).

    PubMed  Google Scholar 

  21. Sun, Z.Y., Kennedy, K.S., Tee, B.C., Damron, J.B. & Allen, M.J. Establishing a critical-size mandibular defect model in growing pigs: characterization of spontaneous healing. J. Oral Maxillofac. Surg. 72, 1852–1868 (2014).

    PubMed  Google Scholar 

  22. Tatara, A.M. et al. Autologously generated tissue-engineered bone flaps for reconstruction of large mandibular defects in an ovine model. Tissue Eng. Part A 21, 1520–1528 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  23. Dard, M. Animal models for experimental surgical research in implant dentistry. in Implant Dentistry Research Guide: Basic, Translational and Experimental Clinical Research. (ed. Ballo, A.) 167–190 (Nova Science Publishers, New York, 2012).

  24. Dard, M. Methods and interpretation of performance studies for dental implants. in Biocompatibility and Performance of Medical Devices (ed. Jean-Pierre Boutrand) 308–344 (Woodhead Publishing, Cambridge, UK, 2012).

    Google Scholar 

  25. Guo, J. et al. Restoration of critical-size defects in the rabbit mandible using porous nanohydroxyapatite-polyamide scaffolds. Tissue Eng. Part A 18, 1239–1252 (2012).

    CAS  PubMed  Google Scholar 

  26. Campillo, V.E., Langonnet, S., Pierrefeu, A. & Chaux-Bodard, A.G. Anatomic and histological study of the rabbit mandible as an experimental model for wound healing and surgical therapies. Lab. Anim. 48, 273–277 (2014).

    CAS  PubMed  Google Scholar 

  27. Miloro, M., Haralson, D.J. & Desa, V. Bone healing in a rabbit mandibular defect using platelet-rich plasma. J. Oral Maxillofac. Surg. 68, 1225–1230 (2010).

    PubMed  Google Scholar 

  28. Rottgers, S.A. et al. Bone morphogenetic protein 2-mediated mandible reconstruction successfully heals bony defects but inhibits concurrent inferior alveolar nerve grafting: a rabbit experimental model. J. Craniofac. Surg. 25, 2241–2245 (2014).

    PubMed  Google Scholar 

  29. Kretlow, J.D. et al. Evaluation of soft tissue coverage over porous polymethylmethacrylate space maintainers within nonhealing alveolar bone defects. Tissue Eng. Part C Methods 16, 1427–1438 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  30. Nguyen, C., Young, S., Kretlow, J.D., Mikos, A.G. & Wong, M. Surface characteristics of biomaterials used for space maintenance in a mandibular defect: a pilot animal study. J. Oral Maxillofac. Surg. 69, 11–18 (2011).

    PubMed  Google Scholar 

  31. Spicer, P.P. et al. In situ formation of porous space maintainers in a composite tissue defect. J. Biomed. Mater. Res. A 100A, 827–833 (2012).

    CAS  Google Scholar 

  32. Petersen, K. et al. Prevention of infections associated with combat-related eye, maxillofacial, and neck injuries. J. Trauma 71, S264–S269 (2011).

    CAS  PubMed  Google Scholar 

  33. Spicer, P.P. et al. Evaluation of antibiotic releasing porous polymethylmethacrylate space maintainers in an infected composite tissue defect model. Acta Biomater. 9, 8832–8839 (2013).

    CAS  PubMed  Google Scholar 

  34. Shah, S.R. et al. Polymer-based local antibiotic delivery for prevention of polymicrobial infection in contaminated mandibular implants. ACS Biomater. Sci. Eng. 2, 558–566 (2016).

    CAS  Google Scholar 

  35. Young, S. et al. Microcomputed tomography characterization of neovascularization in bone tissue engineering applications. Tissue Eng. Part B Rev. 14, 295–306 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  36. Lalani, Z., Wong, M., Brey, E.M., Mikos, A.G. & Duke, P.J. Spatial and temporal localization of transforming growth factor-beta1, bone morphogenetic protein-2, and platelet-derived growth factor-A in healing tooth extraction sockets in a rabbit model. J. Oral Maxillofac. Surg. 61, 1061–1072 (2003).

    PubMed  Google Scholar 

  37. Wen, B. et al. The osseointegration behavior of titanium-zirconium implants in ovariectomized rabbits. Clin. Oral Implants Res. 25, 819–825 (2014).

    PubMed  Google Scholar 

  38. Schon, R., Ohno, K., Kudo, M. & Michi, K. Peri-implant tissue reaction in bone irradiated the fifth day after implantation in rabbits: histologic and histomorphometric measurements. Int. J. Oral Maxillofac. Implants 11, 228–238 (1996).

    CAS  PubMed  Google Scholar 

  39. Jegoux, F. et al. Alveolar ridge augmentation in irradiated rabbit mandibles. J. Biomed. Mater. Res. A 93, 1519–1526 (2010).

    PubMed  Google Scholar 

  40. German, C.L. & Madihally, S.V. Applications of computational modelling and simulation of porous medium in tissue engineering. Computation 4, article 7, http://dx.doi.org/10.3390/computation4010007 (2016).

    Google Scholar 

  41. Collinet-Adler, S., Castro, C.A., Ledonio, C.G., Bechtold, J.E. & Tsukayama, D.T. Acinetobacter baumannii is not associated with osteomyelitis in a rat model: a pilot study. Clin. Orthop. Relat. Res. 469, 274–282 (2011).

    PubMed  Google Scholar 

  42. Vo, T.N., Ekenseair, A.K., Kasper, F.K. & Mikos, A.G. Synthesis, physicochemical characterization, and cytocompatibility of bioresorbable, dual-gelling injectable hydrogels. Biomacromolecules 15, 132–142 (2014).

    CAS  PubMed  Google Scholar 

  43. Sarhaddi, D. et al. Validation of histologic bone analysis following microfil vessel perfusion. J. Histotechnol. 35, 180–183 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  44. Fischer, E.R., Hansen, B.T., Nair, V., Hoyt, F.H. & Dorward, D.W. Scanning electron microscopy. Curr. Protoc. Microbiol. Chapter 2 Unit 2B 2, (2012).

  45. Graham, L. & Orenstein, J.M. Processing tissue and cells for transmission electron microscopy in diagnostic pathology and research. Nat. Protoc. 2, 2439–2450 (2007).

    CAS  PubMed  Google Scholar 

  46. Webster, J.D., Miller, M.A., Dusold, D. & Ramos-Vara, J. Effects of prolonged formalin fixation on diagnostic immunohistochemistry in domestic animals. J. Histochem. Cytochem. 57, 753–761 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  47. Kallai, I. et al. Microcomputed tomography-based structural analysis of various bone tissue regeneration models. Nat. Protoc. 6, 105–110 (2011).

    CAS  PubMed  Google Scholar 

  48. Meganck, J.A., Kozloff, K.M., Thornton, M.M., Broski, S.M. & Goldstein, S.A. Beam hardening artifacts in micro-computed tomography scanning can be reduced by X-ray beam filtration and the resulting images can be used to accurately measure BMD. Bone 45, 1104–1116 (2009).

    PubMed  PubMed Central  Google Scholar 

  49. An, Y.H. & Martin, K.L. Handbook of Histology Methods for Bone and Cartilage (Humana Press, New York, 2003).

  50. Patel, Z.S. et al. Dual delivery of an angiogenic and an osteogenic growth factor for bone regeneration in a critical size defect model. Bone 43, 931–940 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  51. Taschereau, R., Silverman, R.W. & Chatziioannou, A.F. Dual-energy attenuation coefficient decomposition with differential filtration and application to a microCT scanner. Phys. Med. Biol. 55, 1141–1155 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

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Acknowledgements

A.G.M. acknowledges support toward the development of biomaterials for tissue engineering applications by the National Institutes of Health (grant R01 AR068073). A.G.M. and M.E.W. acknowledge support toward the development of materials and techniques through the Armed Forces Institute of Regenerative Medicine (award no. W81XWH-14-2-0004). S.R.S. acknowledges support from a Ruth L. Kirschstein Fellowship from the National Institutes of Health (F30 AR067606). We acknowledge A. Tatara, J. Lam and S. Lu for their assistance with photography, P. Spicer for sharing his experience with the model and S. Frazier for her assistance in providing details of analgesia and intraoperative monitoring.

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Authors and Affiliations

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

    Sarita R Shah & Antonios G Mikos

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

    Simon Young & Mark E Wong

  3. Center for Laboratory Animal Medicine and Care, University of Texas Health Science Center at Houston, Houston, Texas, USA

    Julia L Goldman

  4. Department of Biomaterials, Radboud University Medical Center, Nijmegen, the Netherlands

    John A Jansen

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Contributions

S.R.S. wrote the protocol and developed adaptations to the protocol; S.Y., M.E.W. and A.G.M. developed the protocol; J.L.G. developed the veterinary care plan for the protocol; J.A.J. assisted with the development of analytical protocols.

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Correspondence to Antonios G Mikos.

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Supplementary Notes 1–4 and Supplementary Tables 1 and 2 (PDF 586 kb)

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Shah, S., Young, S., Goldman, J. et al. A composite critical-size rabbit mandibular defect for evaluation of craniofacial tissue regeneration. Nat Protoc 11, 1989–2009 (2016). https://doi.org/10.1038/nprot.2016.122

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