In Vivo 3D Histomorphometry Quantifies Bone Apposition and Skeletal Progenitor Cell Differentiation

Histomorphometry and Micro-CT are commonly used to assess bone remodeling and bone microarchitecture. These approaches typically require separate cohorts of animals to analyze 3D morphological changes and involve time-consuming immunohistochemistry preparation. Intravital Microscopy (IVM) in combination with mouse genetics may represent an attractive option to obtain bone architectural measurements while performing longitudinal monitoring of dynamic cellular processes in vivo. In this study we utilized two-photon, multicolor fluorescence IVM together with a lineage tracing reporter mouse model to image skeletal stem cells (SSCs) in their calvarial suture niche and analyze their differentiation fate after stimulation with an agonist of the canonical Wnt pathway (recombinant Wnt3a). Our in vivo histomorphometry analyses of bone formation, suture volume, and cellular dynamics showed that recombinant Wnt3a induces new bone formation, differentiation and incorporation of SSCs progeny into newly forming bone. IVM technology can therefore provide additional dynamic 3D information to the traditional static 2D histomorphometry.

[1]  Örjan Smedby,et al.  Predicting Trabecular Bone Stiffness from Clinical Cone-Beam CT and HR-pQCT Data; an In Vitro Study Using Finite Element Analysis , 2016, PloS one.

[2]  M. Longaker,et al.  Absence of Endochondral Ossification and Craniosynostosis in Posterior Frontal Cranial Sutures of Axin2−/− Mice , 2013, PloS one.

[3]  Matthew D. Kwan,et al.  Origin Matters: Differences in Embryonic Tissue Origin and Wnt Signaling Determine the Osteogenic Potential and Healing Capacity of Frontal and Parietal Calvarial Bones , 2009, Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research.

[4]  R. Fridman,et al.  Matrix metalloproteinase activity, bone matrix turnover, and tumor cell proliferation in prostate cancer bone metastasis. , 2002, Journal of the National Cancer Institute.

[5]  T. T. Le,et al.  Coherent anti-Stokes Raman scattering imaging of lipids in cancer metastasis , 2009, BMC Cancer.

[6]  Charles P. Lin,et al.  Intravital imaging of osteocytes in mouse calvaria using third harmonic generation microscopy , 2017, PloS one.

[7]  Ralph Weissleder,et al.  Analysis of mitosis and antimitotic drug responses in tumors by in vivo microscopy and single-cell pharmacodynamics. , 2011, Cancer research.

[8]  Ralph Müller,et al.  Volumetric spatial decomposition of trabecular bone into rods and plates--a new method for local bone morphometry. , 2006, Bone.

[9]  Taejun Wang,et al.  In vivo longitudinal visualization of bone marrow engraftment process in mouse calvaria using two-photon microscopy , 2017, Scientific Reports.

[10]  M. Ishii,et al.  Intravital bone imaging by two-photon excitation microscopy to identify osteocytic osteolysis in vivo. , 2015, Bone.

[11]  H. Kronenberg,et al.  Loss of wnt/β‐catenin signaling causes cell fate shift of preosteoblasts from osteoblasts to adipocytes , 2012, Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research.

[12]  P. Schneider,et al.  Techniques to assess bone ultrastructure organization: orientation and arrangement of mineralized collagen fibrils , 2016, Journal of The Royal Society Interface.

[13]  Y. Kraan,et al.  Single-cell Raman and fluorescence microscopy reveal the association of lipid bodies with phagosomes in leukocytes. , 2005, Proceedings of the National Academy of Sciences of the United States of America.

[14]  H Weinans,et al.  Longitudinal micro-CT scans to evaluate bone architecture. , 2005, Journal of musculoskeletal & neuronal interactions.

[15]  Ralph Müller,et al.  Mouse tail vertebrae adapt to cyclic mechanical loading by increasing bone formation rate and decreasing bone resorption rate as shown by time-lapsed in vivo imaging of dynamic bone morphometry. , 2011, Bone.

[16]  Alexander J Makowski,et al.  Predicting mouse vertebra strength with micro-computed tomography-derived finite element analysis. , 2015, BoneKEy reports.

[17]  Charles P. Lin,et al.  Postnatal Calvarial Skeletal Stem Cells Expressing PRX1 Reside Exclusively in the Calvarial Sutures and Are Required for Bone Regeneration , 2017, Stem cell reports.

[18]  T. Foster,et al.  Antibody-labeled fluorescence imaging of dendritic cell populations in vivo. , 2008, Journal of biomedical optics.

[19]  Satoshi Kawata,et al.  Time-lapse Raman imaging of osteoblast differentiation , 2015, Scientific Reports.

[20]  Thomas M. Link,et al.  High-resolution Computed Tomography for Clinical Imaging of Bone Microarchitecture , 2011, Clinical orthopaedics and related research.

[21]  W. Hsu,et al.  Stem cells of the suture mesenchyme in craniofacial bone development, repair and regeneration , 2016, Nature Communications.

[22]  A. Fabre,et al.  Imaging lipid bodies in cells and tissues using third-harmonic generation microscopy , 2005, Nature Methods.

[23]  Johannes E. Schindelin,et al.  Fiji: an open-source platform for biological-image analysis , 2012, Nature Methods.

[24]  Thomas Boudier,et al.  TANGO: a generic tool for high-throughput 3D image analysis for studying nuclear organization , 2013, Bioinform..

[25]  M. Longaker,et al.  Differential activation of canonical Wnt signaling determines cranial sutures fate: a novel mechanism for sagittal suture craniosynostosis. , 2010, Developmental biology.

[26]  A. Dunn,et al.  Influence of optical properties on two-photon fluorescence imaging in turbid samples. , 2000, Applied optics.

[27]  E. Scott,et al.  Extended time-lapse in vivo imaging of tibia bone marrow to visualize dynamic hematopoietic stem cell engraftment , 2016, Leukemia.

[28]  F Meijlink,et al.  Prx1 and Prx2 in skeletogenesis: roles in the craniofacial region, inner ear and limbs. , 1998, Development.

[29]  A. Kawanami,et al.  Prx1 and 3.2kb Col1a1 promoters target distinct bone cell populations in transgenic mice. , 2014, Bone.

[30]  Florent Elefteriou,et al.  Genetic mouse models for bone studies--strengths and limitations. , 2011, Bone.

[31]  I. Veilleux,et al.  In Vivo Cell Tracking With Video Rate Multimodality Laser Scanning Microscopy , 2008, IEEE Journal of Selected Topics in Quantum Electronics.

[32]  T. Ho,et al.  Abstract 5: The Suture Provides a Niche for Mesenchymal Stem Cells of Craniofacial Bones , 2015 .

[33]  Stefan Milz,et al.  Polychrome labeling of bone with seven different fluorochromes: enhancing fluorochrome discrimination by spectral image analysis. , 2005, Bone.

[34]  Raghu Ambekar,et al.  Quantitative second-harmonic generation microscopy for imaging porcine cortical bone: comparison to SEM and its potential to investigate age-related changes. , 2012, Bone.

[35]  J. Helms,et al.  Drugging a Stem Cell Compartment Using Wnt3a Protein as a Therapeutic , 2014, PloS one.

[36]  P. Rüegsegger,et al.  Morphometric analysis of human bone biopsies: a quantitative structural comparison of histological sections and micro-computed tomography. , 1998, Bone.

[37]  Roland Baron,et al.  WNT signaling in bone homeostasis and disease: from human mutations to treatments , 2013, Nature Medicine.

[38]  F. Wise,et al.  In vivo three-photon microscopy of subcortical structures within an intact mouse brain , 2012, Nature Photonics.

[39]  Jiebo Luo,et al.  Spatiotemporal Analyses of Osteogenesis and Angiogenesis via Intravital Imaging in Cranial Bone Defect Repair , 2015, Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research.

[40]  W. Webb,et al.  Nonlinear magic: multiphoton microscopy in the biosciences , 2003, Nature Biotechnology.

[41]  David W. Rowe,et al.  Live-animal tracking of individual haematopoietic stem/progenitor cells in their niche , 2009, Nature.

[42]  B. Tromberg,et al.  Imaging cells and extracellular matrix in vivo by using second-harmonic generation and two-photon excited fluorescence , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[43]  M. Ishii,et al.  Chemorepulsion by blood S1P regulates osteoclast precursor mobilization and bone remodeling in vivo , 2010, The Journal of experimental medicine.

[44]  E. Hirata,et al.  Future Perspective of Single-Molecule FRET Biosensors and Intravital FRET Microscopy. , 2016, Biophysical journal.

[45]  C. P. Winlove,et al.  Structural Analysis of Glycosaminoglycans and Proteoglycans by Means of Raman Microspectrometry , 2009, Connective tissue research.

[46]  J. JEBARAMYAa,et al.  SPECTRAL STUDIES OF TOLUIDINE BLUE O IN THE PRESENCE OF SODIUM DODECYL SULFATE , 2009 .

[47]  Ralph Weissleder,et al.  Intravital Imaging , 2011, Cell.

[48]  M. Ishii,et al.  Quantifying cellular interaction dynamics in 3D fluorescence microscopy data , 2009, Nature Protocols.

[49]  Di Chen,et al.  Anti-DKK1 antibody promotes bone fracture healing through activation of β-catenin signaling. , 2015, Bone.

[50]  R. Baron,et al.  Deletion of a Single Allele of the Dkk1 Gene Leads to an Increase in Bone Formation and Bone Mass , 2006, Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research.

[51]  M. Demay,et al.  Hormonal Regulation of Osteocyte Perilacunar and Canalicular Remodeling in the Hyp Mouse Model of X‐Linked Hypophosphatemia , 2017, Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research.

[52]  V. Nurcombe,et al.  Wnt signaling controls the fate of mesenchymal stem cells. , 2009, Gene.

[53]  M. Braga,et al.  Exploratory Data Analysis , 2018, Encyclopedia of Social Network Analysis and Mining. 2nd Ed..

[54]  M. Ishii,et al.  Dynamic live imaging of bone: opening a new era with ‘bone histodynametry’ , 2013, Journal of Bone and Mineral Metabolism.

[55]  C. Sumen,et al.  ReviewIntravital Microscopy : Visualizing Immunity in Context , 2004 .

[56]  D. Dempster,et al.  Bone histomorphometry: a concise review for endocrinologists and clinicians. , 2010, Arquivos brasileiros de endocrinologia e metabologia.

[57]  Howard I. Maibach,et al.  Non invasive diagnostic techniques in clinical dermatology , 2014 .

[58]  J E Fonseca,et al.  Bone histomorphometry revisited. , 2012, Acta reumatologica portuguesa.

[59]  Sergey Plotnikov,et al.  Second harmonic generation microscopy for quantitative analysis of collagen fibrillar structure , 2012, Nature Protocols.

[60]  Uno Wennergren,et al.  Spatiotemporal Variation in Distance Dependent Animal Movement Contacts: One Size Doesn’t Fit All , 2016, PloS one.

[61]  Sergei A. Vinogradov,et al.  Direct measurement of local oxygen concentration in the bone marrow of live animals , 2014, Nature.

[62]  H. Clevers.  Wnt/beta-catenin signaling in development and disease. , 2006, Cell.

[63]  June-Huyck Hur,et al.  Assessment of Bone Quality using Finite Element Analysis Based upon Micro-CT Images , 2009, Clinics in orthopedic surgery.

[64]  R. Kiesslich,et al.  In vivo histology of Barrett's esophagus and associated neoplasia by confocal laser endomicroscopy. , 2005, Clinical gastroenterology and hepatology : the official clinical practice journal of the American Gastroenterological Association.

[65]  Xunbin Wei,et al.  Imaging Molecular Expression on Vascular Endothelial Cells by In Vivo Immunofluorescence Microscopy , 2006, Molecular imaging.

[66]  M. Ishii,et al.  In vivo fluorescence imaging of bone-resorbing osteoclasts. , 2011, Journal of the American Chemical Society.

[67]  Charles P. Lin,et al.  Endogenous bone marrow MSCs are dynamic, fate-restricted participants in bone maintenance and regeneration. , 2012, Cell stem cell.

[68]  V. Ntziachristos.  Going deeper than microscopy: the optical imaging frontier in biology , 2010, Nature Methods.

[69]  S. Ott,et al.  Histomorphometric measurements of bone turnover, mineralization, and volume. , 2008, Clinical journal of the American Society of Nephrology : CJASN.

[70]  F. Cordelières,et al.  A guided tour into subcellular colocalization analysis in light microscopy , 2006, Journal of microscopy.