title: 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 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.
authors.affiliation: Skeletal Biotech Laboratory, The Hebrew University-Hadassah Faculty of Dental Medicine.
authors.forename: Doron
authors.initials: D
authors.lastname: Cohn Yakubovich
authors.affiliation: Department of Surgery, Cedars-Sinai Medical Center; Board of Governors Regenerative Medicine Institute, Cedars-Sinai Medical Center; Biomedical Imaging Research Institute, Cedars-Sinai Medical Center; wafa.tawackoli@csmc.edu.
authors.forename: Wafa
authors.initials: W
authors.lastname: Tawackoli
authors.affiliation: Department of Surgery, Cedars-Sinai Medical Center; Board of Governors Regenerative Medicine Institute, Cedars-Sinai Medical Center.
authors.forename: Dmitriy
authors.initials: D
authors.lastname: Sheyn
authors.affiliation: Skeletal Biotech Laboratory, The Hebrew University-Hadassah Faculty of Dental Medicine.
authors.forename: Ilan
authors.initials: I
authors.lastname: Kallai
authors.affiliation: Biomedical Imaging Research Institute, Cedars-Sinai Medical Center.
authors.forename: Xiaoyu
authors.initials: X
authors.lastname: Da
authors.affiliation: Skeletal Biotech Laboratory, The Hebrew University-Hadassah Faculty of Dental Medicine; Department of Surgery, Cedars-Sinai Medical Center; Board of Governors Regenerative Medicine Institute, Cedars-Sinai Medical Center; Biomedical Imaging Research Institute, Cedars-Sinai Medical Center.
authors.forename: Gadi
authors.initials: G
authors.lastname: Pelled
authors.affiliation: Skeletal Biotech Laboratory, The Hebrew University-Hadassah Faculty of Dental Medicine; Department of Surgery, Cedars-Sinai Medical Center; Board of Governors Regenerative Medicine Institute, Cedars-Sinai Medical Center; Biomedical Imaging Research Institute, Cedars-Sinai Medical Center.
authors.forename: Dan
authors.initials: D
authors.lastname: Gazit
authors.affiliation: Skeletal Biotech Laboratory, The Hebrew University-Hadassah Faculty of Dental Medicine; Department of Surgery, Cedars-Sinai Medical Center; Board of Governors Regenerative Medicine Institute, Cedars-Sinai Medical Center.
authors.forename: Zulma
authors.initials: Z
authors.lastname: Gazit
mesh_terms: D064591:Allografts / Q000033:anatomy & histology* / Q000098:blood supply / Q000000981:diagnostic imaging; D000818:Animals; D064592:Autografts / Q000098:blood supply / Q000000981:diagnostic imaging; D001861:Bone Regeneration / Q000502:physiology*; D016025:Bone Transplantation / Q000379:methods*; D001842:Bone and Bones / Q000033:anatomy & histology / Q000098:blood supply* / Q000000981:diagnostic imaging; D002454:Cell Differentiation; D005260:Female; D051379:Mice; D008822:Mice, Transgenic; D018919:Neovascularization, Physiologic / Q000502:physiology; D061848:Optical Imaging / Q000379:methods; D010012:Osteogenesis / Q000187:drug effects; D012886:Skull / Q000033:anatomy & histology / Q000098:blood supply / Q000000981:diagnostic imaging / Q000637:transplantation*; D055114:X-Ray Microtomography / Q000379:methods