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

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 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.

Effects of electromagnetic fields treatment on rat critical-sized calvarial defects with a 3D-printed composite scaffold

“…For neovascularization evaluation, six rats from each group were examined using micro-angiography 6 weeks post-surgery. The vascular perfusion was performed using MICROFIL compounds (MV-122, Flow Tech, Carver, MA, USA) via the cardiac approach as mentioned before [29]. Concisely, the rat was anesthetized by intraperitoneal injection of pentobarbital and affixed to the fixation board.…”

PTH-Induced Bone Regeneration and Vascular Modulation Are Both Dependent on Endothelial Signaling

“…The calvarial defect model has been thoroughly described in previous publications [ 15 , 18 , 26 ], including by publication of an instructional video [ 33 ]. Briefly, circular allografts, 4.5 mm in diameter, were harvested from a mouse strain different from the host—C57BL/6 allografts were used in FVB/N hosts, and vice versa.…”

“…The structural analysis of blood vessels was also presented in the video-based protocol mentioned previously [ 33 ]. One week after surgery, the mice were anesthetized by intraperitoneal administration of ketamine/dexmedetomidine, as stated above.…”

Bone-chip system to monitor osteogenic differentiation using optical imaging

“…Longitudinal imaging of the same culture over time can be achieved by the introduction of a genetic tag; one example is the knock-in of the luciferase gene, driven by the osteocalcin promoter, so the bioluminescent signal will reflect osteogenic cell differentiation. This can be performed using transgenic animal model (Ben Arav et al 2012;Cohn Yakubovich et al 2015) when cells are derived from such animals, or by stably transfected primary cells (Sheyn et al 2011). The usage of a constitutively expressed promoter, such as the ubiquitin promotor, presents a reliable tool to monitor cell survival and/or proliferation (Sheyn et al 2011(Sheyn et al , 2016.…”