The authors acknowledge funding from the NIDCR (Grant No. DE019902) and from the Israeli Science Foundation (Grant No. 382/13).

The protocol follows the guidelines of the institutional animal care and use committee (IACUC) of The Hebrew University of Jerusalem, Israel (Request No. MD-12-13524-4), an AAALAC approved facility, and by the Cedars-Sinai Medical Center IACUC (Request No. 3770). The animals were treated in strict adherence to NIH guidelines.
1. Preparation of Bone Allografts
<ol>
	<li>Euthanize 7- to 8-week-old Balb/C mice, or any strain different from the recipient, using the standard method of CO2</s...

<ol>
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The authors have nothing to disclose.

The aim of the multimodal imaging approaches described here is to enable meticulous investigation of the angiogenesis-osteogenesis axis in the context of cranial bone grafting. Neovascularization was imaged using a &#181;CT protocol, which allowed an accurate high-resolution 3D demonstration of the vascular tree feeding the entire cranial defect. &#181;CT data can be readily analyzed using advanced tools such as IPL software. For example, the thickness analysis shown in Figure 3C revealed that the main d...

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 angiogenesis2,3. 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 tree can be investigated by histological analysis. Since histology relies on sectioning tissue, there is a high probability that the resulting image will be distorted. To address this problem, intravital microscopy can be performed to image intact blood vessels4; however, this method is limited to one-plane imaging. &#181;CT scanning of specimens obtained from an animal perfused with contrast agent allows 3D imaging of the vascular network that feeds the regeneration site5. This approach allows a highly detailed demonstration of an organ&#8217;s vasculature as a whole, as well as a meticulous analysis of blood vessel distribution. Furthermore, &#181;CT enables differentiation between varied diameters of blood vessels, which characterize the different subtypes of blood vessels.
We hypothesized that implantation of a calvarial autograft will induce greater neovascularization than implantation of an allograft, and this increased neovascularization will lead, in turn, to enhanced bone formation.To pursue this hypothesis we employed a variety of techniques. We investigated patterns of the newly formed vascular tree by performing a &#181;CT-based analysis. We measured blood perfusion using a blood-pool fluorescent probe. Next, we assessed bone tissue mineralization by FLI of a hydroxyapatite-directed probe and &#181;CT analysis. Finally, we monitored stem cell recruitment and differentiation, performing BLI in transgenic mice in which luciferase is expressed in osteocalcin-positive cells.

<table border="0" cellpadding="0" cellspacing="0" width="614">
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</table>

computed tomography and optical imaging of osteogenesis-angiogenesis coupling to assess integration of cranial bone autografts and allografts

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&#8211;computed tomography (&#181;CT) 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 &#181;CT 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, &#181;CT 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 7th 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 7th and 10th 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.

Neovascularization was assessed by &#181;CT volumetric analysis and by FLI using a fluorescent blood-borne agent to quantify blood perfusion. Seven days after surgery, &#181;CT scanning demonstrated a significantly higher volume of small- and medium-diameter blood vessels in mice that had received autografts than in mice that had received allografts harvested from C57BL/6&#160;(Figure 3A). Interestingly, in the autograft group the newly formed vascular tree appeared to reach the entire defect area, where...

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

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.

A major parameter determining the success of a bone-grafting procedure is vascularization of the area surrounding the graft. We hypothesized that ...

computed-tomography-optical-imaging-osteogenesis-angiogenesis

Research

JoVE Journal

Bioengineering

11.3K Görüntüleme.  The Hebrew University–Hadassah Faculty of Dental Medicine.  Otolog ve allojenik kemik greft implantasyonu önemli kraniofasial kemik kaybını tedavi yaklaşımları kabul teşkil. Oysa neovaskülarizasyon, hücre farklılaşması ve kemik oluşumu arasındaki etkileşime greft bileşiminin etkisi belirsizdir. Biz greft yakınlık anjiyogenez-osteogenesis bağımlılığın aydınlatmak için amaçlandığı multimodal görüntüleme protokol mevcut. 