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      An Ectopic Imaging Window for Intravital Imaging of Engineered Bone Tissue

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          ABSTRACT

          Tissue engineering is a promising branch of regenerative medicine, but its clinical application remains limited because thorough knowledge of the in vivo repair processes in these engineered implants is limited. Common techniques to study the different phases of bone repair in mice are destructive and thus not optimal to gain insight into the dynamics of this process. Instead, multiphoton‐intravital microscopy (MP‐IVM) allows visualization of (sub)cellular processes at high resolution and frequency over extended periods of time when combined with an imaging window that permits optical access to implants in vivo. In this study, we have developed and validated an ectopic imaging window that can be placed over a tissue‐engineered construct implanted in mice. This approach did not interfere with the biological processes of bone regeneration taking place in these implants, as evidenced by histological and micro–computed tomography (μCT)‐based comparison to control ectopic implants. The ectopic imaging window permitted tracking of individual cells over several days in vivo. Furthermore, the use of fluorescent reporters allowed visualization of the onset of angiogenesis and osteogenesis in these constructs. Taken together, this novel imaging window will facilitate further analysis of the spatiotemporal regulation of cellular processes in bone tissue–engineered implants and provides a powerful tool to enhance the therapeutic potential of bone tissue engineering. © 2017 The Authors JBMR Plus published by Wiley Periodicals, Inc. on behalf of American Society for Bone and Mineral Research.

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          Most cited references30

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          NIH Image to ImageJ: 25 years of image analysis.

          For the past 25 years NIH Image and ImageJ software have been pioneers as open tools for the analysis of scientific images. We discuss the origins, challenges and solutions of these two programs, and how their history can serve to advise and inform other software projects.
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            Osteoblast precursors, but not mature osteoblasts, move into developing and fractured bones along with invading blood vessels.

            During endochondral bone development, the first osteoblasts differentiate in the perichondrium surrounding avascular cartilaginous rudiments; the source of trabecular osteoblasts inside the later bone is, however, unknown. Here, we generated tamoxifen-inducible transgenic mice bred to Rosa26R-LacZ reporter mice to follow the fates of stage-selective subsets of osteoblast lineage cells. Pulse-chase studies showed that osterix-expressing osteoblast precursors, labeled in the perichondrium prior to vascular invasion of the cartilage, give rise to trabecular osteoblasts, osteocytes, and stromal cells inside the developing bone. Throughout the translocation, some precursors were found to intimately associate with invading blood vessels, in pericyte-like fashion. A similar coinvasion occurs during endochondral healing of bone fractures. In contrast, perichondrial mature osteoblasts did not exhibit perivascular localization and remained in the outer cortex of developing bones. These findings reveal the specific involvement of immature osteoblast precursors in the coupled vascular and osteogenic transformation essential to endochondral bone development and repair. 2010 Elsevier Inc. All rights reserved.
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              Fracture healing as a post-natal developmental process: molecular, spatial, and temporal aspects of its regulation.

              Fracture healing is a specialized post-natal repair process that recapitulates aspects of embryological skeletal development. While many of the molecular mechanisms that control cellular differentiation and growth during embryogenesis recur during fracture healing, these processes take place in a post-natal environment that is unique and distinct from those which exist during embryogenesis. This Prospect Article will highlight a number of central biological processes that are believed to be crucial in the embryonic differentiation and growth of skeletal tissues and review the functional role of these processes during fracture healing. Specific aspects of fracture healing that will be considered in relation to embryological development are: (1) the anatomic structure of the fracture callus as it evolves during healing; (2) the origins of stem cells and morphogenetic signals that facilitate the repair process; (3) the role of the biomechanical environment in controlling cellular differentiation during repair; (4) the role of three key groups of soluble factors, pro-inflammatory cytokines, the TGF-beta superfamily, and angiogenic factors, during repair; and (5) the relationship of the genetic components that control bone mass and remodeling to the mechanisms that control skeletal tissue repair in response to fracture. Copyright 2003 Wiley-Liss, Inc.
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                Author and article information

                Contributors
                geert.carmeliet@kuleuven.be
                Journal
                JBMR Plus
                JBMR Plus
                10.1002/(ISSN)2473-4039
                JBM4
                JBMR Plus
                John Wiley and Sons Inc. (Hoboken )
                2473-4039
                31 January 2018
                March 2018
                : 2
                : 2 ( doiID: 10.1002/jbm4.v2.2 )
                : 92-102
                Affiliations
                [ 1 ] Laboratory of Clinical and Experimental Endocrinology Department of Chronic Diseases, Metabolism and Ageing KU Leuven Leuven Belgium
                [ 2 ] Prometheus Division of Skeletal Tissue Engineering KU Leuven Leuven Belgium
                Author notes
                [*] [* ] Address correspondence to: Geert Carmeliet, MD, PhD, Laboratory of Clinical and Experimental Endocrinology, Herestraat 49 bus 902, 3000 Leuven, Belgium. E‐mail: geert.carmeliet@ 123456kuleuven.be

                Article
                JBM410028
                10.1002/jbm4.10028
                6124161
                e6cca284-92ce-4835-9177-61d717f300cd
                © 2017 The Authors JBMR Plus published by Wiley Periodicals, Inc. on behalf of American Society for Bone and Mineral Research.

                This is an open access article under the terms of the http://creativecommons.org/licenses/by/4.0/ License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.

                History
                : 11 July 2017
                : 11 December 2017
                : 12 December 2017
                Page count
                Figures: 5, Tables: 0, Pages: 11, Words: 6154
                Funding
                Funded by: Scientific Research‐Flanders
                Award ID: G.0A7213 and G.096414
                Funded by: BOF‐KU Leuven GOA
                Award ID: 3M120209
                Funded by: Hercules Foundation
                Award ID: AKUL/11/033
                Categories
                Original Article
                Original Articles
                Custom metadata
                2.0
                jbm410028
                March 2018
                Converter:WILEY_ML3GV2_TO_NLMPMC version:version=5.4.7.1 mode:remove_FC converted:05.09.2018

                analysis/quantitation of bone,bioengineering,implants

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