Development Of A Novel Murine Distraction Device To Investigate Bone Regeneration In Long Bone Distraction Osteogenesis
Harsh N. Shah, MPH, Ankit Salhotra, B.S., Courtney A. Stockman, B.S., Derrick C. Wan, M.D., Michael T. Longaker, M.D.,M.B.A.
Stanford University, Stanford, CA, USA.
PURPOSE: Distraction osteogenesis (DO) promotes endogenous bone formation across a mechanically controlled environment, providing anatomical and functional replacement of deficient tissue. The application of DO, to the appendicular skeleton, has revolutionized the treatment of many congenital and acquired defects. Here, we describe the development of a novel mouse distraction model for the tibia.
METHODS: Tibial distraction devices were manufactured using computer-aided design (CAD) software (SolidWorks) and 3D-printing (AW3D AXIOM 3D Printer). One 0.6 mm hole was drilled 3mm anterior and one 3mm posterior to a line dividing the tibial crest. An osteotomy was performed at the tibial crest using a diamond disc saw (Brasseler, Inc.). Distraction plates were secured with insertion of tight fit 0.65 mm screws (McMaster-Carr). Animals were divided into four groups: sham (exposure of the tibia and device placement without osteotomy), fracture (osteotomy without distraction), acutely lengthened, and gradually distracted. The gradual distraction protocol consisted of a 5-day latency period after the initial osteotomy and fixation of the distraction device, followed by 10 days of distraction at a rate of 0.15 mm every 12 hours, and 28 days of bone consolidation and remodeling. For our acute lengthening protocol, a 3.0 mm lengthening was performed following a 5-day latency period, with a consolidation period ending at 43 days post-operation (Figure 1A).
RESULTS: Bone successfully regenerated within the surgically created gap using the novel distraction device. Micro computed tomography (CT) images of the sham group presented native, unperturbed bone, while the acute lengthening group images showed the absence of bone regeneration at the site of the osteotomy (Figure 1B). Bone regeneration occurred in the fracture group and the gradual distraction group. Upon quantitative analysis, the bone volume per tissue volume (BV/TV) (***P<0.001) and callus volume (CV) (****P<0.0001) were significantly higher in the distraction group compared to the sham group (Figure 1C). Finally, histological staining through Movat Pentachrome determine the tissue types present within the gap of the four groups. Pentachrome staining of the acute group stained for fibrous tissue at the site of the osteotomy. Pentachrome staining of the fracture group indicated an overt presence of cartilage, and the staining in the distraction group showed the formation of new bone (Figure 1D).
CONCLUSIONS: We have developed a new model for long bone distraction osteogenesis in the mouse. Future work will include applying this model to investigate the mechanisms underlying bone regeneration, and potential therapeutics that can quicken regeneration.
Figure 1. (A) Timeline of the surgical manipulation for the four groups. (B) Micro-computed tomography images of the tibia at post-operative day (POD) 43 for each group. Box indicates the osteotomy site. (C) Quantification of bone volume per tissue volume (BV/TV) and callus volume (CV) from the micro-CT images (*P<0.05, ** P<0.01, *** P<0.001, **** P<0.0001, n = 5; Studentís t test). (D) Movat Pentachrome staining. The white-dotted box represents area of new tissue formation.
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