One of the greatest enigmas of modern biology is how the geometry of muscular and skeletal structures are created and how their development is controlled during growth and regeneration. Scaling and shaping of vertebrate muscles and skeletal elements has always been enigmatic and required an advanced technical level in order to analyse the cell distribution in 3D. In this work, synchrotron X-ray computed microtomography (µCT) and chemical contrasting has been exploited for a quantitative analysis of the 3D-cell distribution in tissues of a developing salamander (Pleurodeles waltl) limb - a key model organism for vertebrate regeneration studies. We mapped the limb muscles, their size and shape as well as the number and density of cells within the extracellular matrix of the developing cartilage. By using tomographic approach, we explored the polarity of the cells in 3D, in relation to the structure of developing joints. We found that the polarity of chondrocytes correlates with the planes in joint surfaces and also changes along the length of the cartilaginous elements. Our approach generates data for the precise computer simulations of muscle-skeletal regeneration using cell dynamics models, which is necessary for the understanding how anisotropic growth results in the precise shapes of skeletal structures.

Central European Institute of Technology Brno University of Technology Brno Czech Republic

Department of Cellular and Molecular Biology Karolinska Institutet Solna 171777 Stockholm Sweden

Department of Molecular Neurosciences Medical University Vienna Vienna Austria

Department of Orthopaedics Xiangya Hospital Central South University Changsha Hunan Province China

Department of Physiology and Pharmacology Karolinska Institutet Solna 171777 Stockholm Sweden

Elettra Sincrotrone Trieste S C p A Basovizza Trieste Italy

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Sci Rep. 2020 Feb 5;10(1):2276. doi: 10.1038/s41598-020-59021-3. PubMed

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Graf BW, Boppart SA. Imaging and Analysis of Three-Dimensional Cell Culture Models. Methods Mol Biol. 2010;591:211–227. doi: 10.1007/978-1-60761-404-3_13. PubMed DOI PMC

Hedlund H, Brismar H, Widholm M, Svensson O. Studies of cell columns of articular cartilage using UV-Confocal scanning laser microscopy and 3D image processing. ‎J. Musculoskelet. Res. 1994;3:93–98.

Artym Vira V., Matsumoto Kazue. Imaging Cells in Three-Dimensional Collagen Matrix. Current Protocols in Cell Biology. 2010;48(1):10.18.1-10.18.20. doi: 10.1002/0471143030.cb1018s48. PubMed DOI PMC

Morgan F, Barbarese E, Carson JH. Visualizing cells in three dimensions using confocal microscopy, image reconstruction and isosurface rendering: application to glial cells in mouse central nervous system. Scanning Microsc. 1992;6:345–56. PubMed

Pawley, J. Handbook of Biological Confocal Microscopy. (Springer, 1995).

Reihani SNS, Oddershede LB. Confocal microscopy of thick specimens. J Biomed. Opt. 2009;14:030513. doi: 10.1117/1.3156813. PubMed DOI

Ewald AJ, Mcbride H, Reddington M, Fraser SE, Kerschmann R. Surface imaging microscopy, an automated method for visualizing whole embryo samples in three dimensions at high resolution. Dev Dyn. 2002;225:369–375. doi: 10.1002/dvdy.10169. PubMed DOI

Helmchen F, Denk W. Deep tissue two-photon microscopy. Nat. Methods. 2005;2:932–940. doi: 10.1038/nmeth818. PubMed DOI

Ustione A, Piston DW. A simple introduction to multiphoton microscopy: A simple introduction to multiphoton microscopy. J Microsc. 2011;243:221–226. doi: 10.1111/j.1365-2818.2011.03532.x. PubMed DOI

Schmitt JM. Optical coherence tomography (OCT): a review. IEEE J. Quantum Electron. 1999;5:1205–1215. doi: 10.1109/2944.796348. DOI

Fercher AF, Drexler W, Hitzenberger CK, Lasser T. Optical coherence tomography-principles and applications. Rep.Prog. Phys. 2003;66:239. doi: 10.1088/0034-4885/66/2/204. DOI

Campbell JP, et al. Detailed Vascular Anatomy of the Human Retina by Projection-Resolved Optical Coherence Tomography Angiography. Sci. Rep. 2017;7:42201. doi: 10.1038/srep42201. PubMed DOI PMC

Le Gros MA, McDermott G, Larabell CA. X-ray tomography of whole cells. Curr Opin Struct Biol. 2005;15:593–600. doi: 10.1016/j.sbi.2005.08.008. PubMed DOI

Larabell CA, Nugent KA. Imaging cellular architecture with X-rays. Curr Opin Struct Biol. 2010;20:623–631. doi: 10.1016/j.sbi.2010.08.008. PubMed DOI PMC

Langer, M. et al. Assessment of imaging quality in magnified phase CT of human bone tissue at the nanoscale. Proc.SPIE. 10391, 10.1117/12.2272561 (SPIE, 2017).

Attwood, D. T. Soft X-rays and extreme ultraviolet radiation: Principles and application. (Cambridge University press, 1999).

Krenkel M, et al. Phase-contrast zoom tomography reveals precise locations of macrophages in mouse lungs. Sci. Rep. 2015;5:9973. doi: 10.1038/srep09973. PubMed DOI PMC

Snigirev A, Snigireva I, Kohn V, Kuznetsov S, Schelokov I. On the possibilities of x‐ray phase contrast microimaging by coherent high‐energy synchrotron radiation. Rev. Sci. Instrum. 1995;66:5486–5492. doi: 10.1063/1.1146073. DOI

Wilkins SW, Gureyev TE, Gao D, Pogany A, Stevenson AW. Phase-contrast imaging using polychromatic hard X-rays. Nature. 1996;384:335. doi: 10.1038/384335a0. DOI

Baruchel, J., Buffiére, J. Y., Maire, E., Merle, P. & Peix, G. X-ray tomography in material science, general principles. (Hermes Science Publications, 2000).

Cloetens P, Barrett R, Baruchel J, Guigay JP, Schlenker M. Phase objects in synchrotron radiation hard X-ray imaging. J PhysD Appl Phys. 1996;29:133–146. doi: 10.1088/0022-3727/29/1/023. DOI

Kaiser J, et al. Investigation of the microstructure and mineralogical composition of urinary calculi fragments by synchrotron radiation X-ray microtomography: a feasibility study. Urol Res. 2011;39:259–267. doi: 10.1007/s00240-010-0343-9. PubMed DOI

Astolfo A, et al. In vivo visualization of gold-loaded cells in mice using x-ray computed tomography. Nanomedicine. 2013;9:284–292. doi: 10.1016/j.nano.2012.06.004. PubMed DOI

Larsson, D. H., Vågberg, W., Yaroshenko, A., Yildirim, A. Ö. & Hertz, H. M. High-resolution short-exposure small-animal laboratory x-ray phase-contrast tomography. Sci. Rep. 6, 10.1038/srep39074 (2016). PubMed PMC

Dullin C, et al. μCT of ex-vivo stained mouse hearts and embryos enables a precise match between 3D virtual histology, classical histology and immunochemistry. PloS one. 2017;12:e0170597. doi: 10.1371/journal.pone. PubMed DOI PMC

Saccomano Mara, Albers Jonas, Tromba Giuliana, Dobrivojević Radmilović Marina, Gajović Srećko, Alves Frauke, Dullin Christian. Synchrotron inline phase contrast µCT enables detailed virtual histology of embedded soft-tissue samples with and without staining. Journal of Synchrotron Radiation. 2018;25(4):1153–1161. doi: 10.1107/S1600577518005489. PubMed DOI

Albers J., Pacilé S., Markus M. A., Wiart M., Vande Velde G., Tromba G., Dullin C. X-ray-Based 3D Virtual Histology—Adding the Next Dimension to Histological Analysis. Molecular Imaging and Biology. 2018;20(5):732–741. doi: 10.1007/s11307-018-1246-3. PubMed DOI

Albers J. et al. X-ray based virtual histology allows guided sectioning of heavy ion stained murine lungs for histological analysis. Sci. Rep. 8, 10.1038/s41598-018-26086-0 (2018). PubMed PMC

Momose, A., Takeda, T., Itaj, Y. & Hirano, K. Phase−contrast X−ray computed tomography for observing biological soft tissues. Nat. Med. 2 (1996). PubMed

Beltran MA, Paganin DM, Uesugi K, Kitchen MJ. 2D and 3D X-ray phase retrieval of multi-material objects using a single defocus distance. Opt Express. 2010;18:6423–36. doi: 10.1364/OE.18.006423. PubMed DOI

Metscher BD. MicroCT for comparative morphology: simple staining methods allow high-contrast 3D imaging of diverse non-mineralized animal tissues. BMC Physiol. 2009;9:11. doi: 10.1186/1472-6793-9-11. PubMed DOI PMC

Metscher Brian D. MicroCT for developmental biology: A versatile tool for high-contrast 3D imaging at histological resolutions. Developmental Dynamics. 2009;238(3):632–640. doi: 10.1002/dvdy.21857. PubMed DOI

Shinohara M, et al. Atherosclerotic plaque imaging using phase-contrast X-ray computed tomography. Am J Physiol Heart Circ Physiol. 2008;294:H1094–H1100. doi: 10.1152/ajpheart.01149.2007. PubMed DOI

Zanette I, et al. Holotomography versus X-ray grating interferometry: A comparative study. Applied Physics Letters. 2013;103:244105. doi: 10.1063/1.4848595. DOI

Lang S, et al. Experimental comparison of grating- and propagation-based hard X-ray phase tomography of soft tissue. Journal of Applied Physics. 2014;116:154903. doi: 10.1152/ajpheart.01149.2007. DOI

Kumar A. et al. An orphan gene is necessary for preaxial digit formation during salamander limb development. Nat Communn. 610.1038/ncomms9684 (2015). PubMed PMC

Frobisch NB, Shubin NH. Salamander limb development: integrating genes, morphology, and fossils. Dev. Dyn. 2011;240:1087–1099. doi: 10.1002/dvdy.22629. PubMed DOI

Godwin JW, et al. Macrophages are required for adult salamander limb regeneration. Proc Natl Acad Sci USA. 2013;23:9415–20. doi: 10.1073/pnas.1300290110. PubMed DOI PMC

Yun MH, et al. Regulation of p53 is critical for vertebrate limb regeneration. Proc Natl Acad Sci USA. 2013;43:17392–7. doi: 10.1073/pnas.1310519110. PubMed DOI PMC

Cosden RS, et al. Intrinsic repair of full-thickness articular cartilage defects in the axolotl salamander. Osteoarthritis Cartilage. 2011;19:200–205. doi: 10.1016/j.joca.2010.11.005. PubMed DOI PMC

Khan IM, et al. The development of synovial joints. Curr. Top. Dev. Biol. 2007;79:1–36. doi: 10.1016/S0070-2153(06)79001-9. PubMed DOI

Cosden-Decker RS, et al. Structural and functional analysis of intra-articular interzone tissue in axolotl salamanders. Osteoarthritis Cartilage. 2012;20:1347–1356. doi: 10.1016/j.joca.2012.07.002. PubMed DOI PMC

Adameyko I, Fried K. The Nervous System Orchestrates and Integrates Craniofacial Development: A Review. Front. Physiol. 2016;7:49. doi: 10.3389/fphys.2016.00049. PubMed DOI PMC

Davidson LA. Epithelial machines that shape the embryo. Trends Cell Biol. 2012;22:82–87. doi: 10.1016/j.tcb.2011.10.005. PubMed DOI PMC

Boehm B, et al. The Role of Spatially Controlled Cell Proliferation in Limb Bud Morphogenesis. PLoS Biol. 2010;8:e1000420. doi: 10.1371/journal.pbio.1000420. PubMed DOI PMC

Abzhanov A, Tabin CJ. Shh and Fgf8 act synergistically to drive cartilage outgrowth during cranial development. Dev Biol. 2004;273:134–148. doi: 10.1016/j.ydbio.2004.05.028. PubMed DOI

Gros J, Tabin CJ. Vertebrate Limb Bud Formation Is Initiated by Localized Epithelial-to-Mesenchymal Transition. Science. 2014;343:1253–1256. doi: 10.1126/science.1248228. PubMed DOI PMC

Kaucka M, et al. Oriented clonal cell dynamics enables accurate growth and shaping of vertebrate cartilage. Elife. 2017;6:e25902. doi: 10.7554/eLife.25902. PubMed DOI PMC

Li L, et al. Superficial cells are self-renewing chondrocyte progenitors, which form the articular cartilage in juvenile mice. FASEB J. 2017;31:1067–1084. doi: 10.1096/fj.201600918R. PubMed DOI PMC

Kaucka M, et al. Signals from the brain and olfactory epithelium control shaping of the mammalian nasal capsule cartilage. eLife eLife. 2018;7:e34465. doi: 10.7554/eLife.34465. PubMed DOI PMC

Buckingham M, et al. The formation of skeletal muscle: from somite to limb. J Anat. 2003;202:59–68. doi: 10.1046/j.1469-7580.2003.00139.x. PubMed DOI PMC

Stockdale FE, et al. Molecular and cellular biology of avian somite development. Dev Dyn. 2000;219:304–21. doi: 10.1002/1097-0177(2000)9999:9999<::AID-DVDY1057>3.0.CO;2-5. PubMed DOI

Roddy KA, et al. Mechanical Influences on Morphogenesis of the Knee Joint Revealed through Morphological, Molecular and Computational Analysis of Immobilised Embryos. PLoS One. 2011;6:e17526. doi: 10.1371/journal.pone.0017526. PubMed DOI PMC

Rapaport D, et al. Short stature in Duchenne muscular dystrophy. Growth Regul. 1991;1:11–5. PubMed

Cairns DM, et al. The role of muscle cells in regulating cartilage matrix production. J Orthop Res. 2010;28:529–536. PubMed PMC

Zehbe R, et al. Characterization of oriented protein-ceramic and protein-polymer-composites for cartilage tissue engineering using synchrotron m-CT. Int. J. Mater. Res. 2007;98:562–568. doi: 10.3139/146.101509. DOI

Zehbe R, et al. Going beyond histology. Synchrotron micro-computed tomography as a methodology for biological tissue characterization: from tissue morphology to individual cells. J R Soc Interface. 2010;7:49–59. doi: 10.1098/rsif.2008.0539. PubMed DOI PMC

Hieber S, et al. Tomographic brain imaging with nucleolar detail and automatic cell counting. Sci. Rep. 2016;6:32156. doi: 10.1038/srep32156. PubMed DOI PMC

Elewa A, et al. Reading and editing the Pleurodeles waltl genome reveals novel features of tetrapod regeneration. Nat. Communn. 2017;8:9. doi: 10.1038/s41467-017-01964-. PubMed DOI PMC

Tesařová M, et al. Use of micro computed-tomography and 3D printing for reverse engineering of mouse embryo nasal capsule. JINST. 2016;11:C03006–C03006. doi: 10.1088/1748-0221/11/03/C03006. DOI

Constantine VS, Mowry RW. Selective staining of human dermal collagen. J. Invest Dermatol. 1968;50:414–418. doi: 10.1038/jid.1968.67. PubMed DOI

Nemetschek T, Riedl H, Jonak R. Topochemistry of the binding of phosphotungstic acid to collagen. J. Mol. Biol. 1979;133:67–83. doi: 10.1016/0022-2836(79)90251-1. PubMed DOI

Adams R, Bischof L. Seeded region growing. IEEE Trans. Pattern Anal. Mach. Intell. 1994;16:641–647. doi: 10.1109/34.295913. DOI

Schneider CA, Rasband WS, Eliceiri KW. NIH Image to ImageJ: 25 years of image analysis. Nat. methods. 2012;9:671. doi: 10.1038/nmeth.2089. PubMed DOI PMC

Boudier T. Elaboration d’un modèle de déformation pour la détection de contours aux formes complexes. Innov. Techn. Biol. Med. 1997;18:1.

Andrey, P. & Boudier, T. Adaptive active contours (snakes) for the segmentation of complex structures in biological images. in Centre de Recherche Public Henri Tudor Copyright Notice 181 (2006).

Brun F, et al. Pore3D: A software library for quantitative analysis of porous media. Nucl. Instr. Meth. Phys. Res. 2010;615:326–332. doi: 10.1016/j.nima.2010.02.063. DOI

Hartigan JA, Wong MA. Algorithm AS 136: A K-Means Clustering Algorithm. Applied Statistics. 1979;28:100. doi: 10.2307/2346830. DOI

Zandomeneghi D, et al. Quantitative analysis of X-ray microtomography images of geomaterials: Application to volcanic rocks. Geosphere. 2010;6:793–804. doi: 10.1130/GES00561.1. DOI

Viani A, et al. Microstructural characterization of dental zinc phosphate cements using combined small angle neutron scattering and microfocus X-ray computed tomography. Dent Mater. 2017;4:402–417. doi: 10.1016/j.dental.2017.01.008. PubMed DOI

Wong M, et al. Zone-specific cell biosynthetic activity in mature bovine articular cartilage: a new method using confocal microscopic stereology and quantitative autoradiography. J. Orthop. Res. 1996;14:424–432. doi: 10.1002/jor.1100140313. PubMed DOI

Jadin KD, et al. Depth-varying density and organization of chondrocytes in immature and mature bovine articular cartilage assessed by 3D imaging and analysis. J. Histochem. Cytochem. 2005;53:1109–1119. doi: 10.1369/jhc.4A6511.2005. PubMed DOI

Brunt LH, et al. Finite element modelling predicts changes in joint shape and cell behaviour due to loss of muscle strain in jaw development. J Biochem. 2015;48:3112–22. doi: 10.1016/j.jbiomech.2015.07.017. PubMed DOI PMC

Kaucka M, et al. Analysis of neural crest–derived clones reveals novel aspects of facial development. Science Advances. 2016;2:e1600060. doi: 10.1126/sciadv.1600060. PubMed DOI PMC

Joven Alberto, Kirkham Matthew, Simon András. Methods in Molecular Biology. New York, NY: Springer New York; 2015. Husbandry of Spanish Ribbed Newts (Pleurodeles waltl) pp. 47–70. PubMed

Brun F, et al. Enhanced and Flexible Software Tools for X-ray Computed Tomography at the Italian Synchrotron Radiation Facility Elettra. Fundamenta Informaticae. 2015;141:233–243. doi: 10.3233/FI-2015-1273. DOI

Altered developmental programs and oriented cell divisions lead to bulky bones during salamander limb regeneration

Living in darkness: Exploring adaptation of Proteus anguinus in 3 dimensions by X-ray imaging

An interactive and intuitive visualisation method for X-ray computed tomography data of biological samples in 3D Portable Document Format