Computed Tomography and Optical Imaging of Osteogenesis-angiogenesis Coupling to Assess Integration of Cranial Bone Autografts and Allografts

Doron Cohn Yakubovich; Wafa Tawackoli; Dmitriy Sheyn; Ilan Kallai; Xiaoyu Da; Gadi Pelled; Dan Gazit; Zulma Gazit

doi:10.3791/53459

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Method Article

Computed Tomography and Optical Imaging of Osteogenesis-angiogenesis Coupling to Assess Integration of Cranial Bone Autografts and Allografts

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DOI:

10.3791/53459

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December 22nd, 2015

Doron Cohn Yakubovich¹ , Wafa Tawackoli²^,³^,⁴ , Dmitriy Sheyn²^,³ , Ilan Kallai¹ , Xiaoyu Da⁴ , Gadi Pelled¹^,²^,³^,⁴ , Dan Gazit¹^,²^,³^,⁴ , Zulma Gazit¹^,²^,³

¹Skeletal Biotech Laboratory, The Hebrew University–Hadassah Faculty of Dental Medicine, ²Department of Surgery, Cedars-Sinai Medical Center, ³Board of Governors Regenerative Medicine Institute, Cedars-Sinai Medical Center, ⁴Biomedical Imaging Research Institute, Cedars-Sinai Medical Center

Summary

Implantation of autologous and allogeneic bone grafts constitute accepted approaches to treat major craniofacial bone loss. Yet the effect of graft composition on the interplay among neovascularization, cell differentiation and bone formation is unclear. We present a multimodal imaging protocol aimed to elucidate the angiogenesis-osteogenesis interdependence at the graft proximity.

Abstract

A major parameter determining the success of a bone-grafting procedure is vascularization of the area surrounding the graft. We hypothesized that implantation of a bone autograft would induce greater bone regeneration by abundant blood vessel formation. To investigate the effect of the graft on neovascularization at the defect site, we developed a micro–computed tomography (µCT) approach to characterize newly forming blood vessels, which involves systemic perfusion of the animal with a polymerizing contrast agent. This method enables detailed vascular analysis of an organ in its entirety. Additionally, blood perfusion was assessed using fluorescence imaging (FLI) of a blood-borne fluorescent agent. Bone formation was quantified by FLI using a hydroxyapatite-targeted probe and µCT analysis. Stem cell recruitment was monitored by bioluminescence imaging (BLI) of transgenic mice that express luciferase under the control of the osteocalcin promoter. Here we describe and demonstrate preparation of the allograft, calvarial defect surgery, µCT scanning protocols for the neovascularization study and bone formation analysis (including the in vivo perfusion of contrast agent), and the protocol for data analysis.

The 3D high-resolution analysis of vasculature demonstrated significantly greater angiogenesis in animals with implanted autografts, especially with respect to arteriole formation. Accordingly, blood perfusion was significantly higher in the autograft group by the 7^th day after surgery. We observed superior bone mineralization and measured greater bone formation in animals that received autografts. Autograft implantation induced resident stem cell recruitment to the graft-host bone suture, where the cells differentiated into bone-forming cells between the 7^th and 10^th postoperative day. This finding means that enhanced bone formation may be attributed to the augmented vascular feeding that characterizes autograft implantation. The methods depicted may serve as an optimal tool to study bone regeneration in terms of tightly bounded bone formation and neovascularization.

Introduction

Craniofacial bone loss due to trauma, tumor resection, decompressive craniotomy, and congenital defect rarely heals by itself and presents a clear unmet clinical need. Autologous bone grafts and allogeneic bone grafts are extensively used to treat these conditions¹.

It is widely accepted that osteogenesis is tightly coupled with angiogenesis^2,3. Thus, the complete study of a proposed therapy for bone regeneration should include a comprehensive investigation of the vascular tree forming throughout the entire defect site. There are several available methods to characterize vascularization in research models. The vascular....