Skip to main content

Thank you for visiting You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

A network of trans-cortical capillaries as mainstay for blood circulation in long bones


Closed circulatory systems underlie the function of vertebrate organs, but in long bones their structure is unclear although they constitute the exit route for bone marrow (BM) leukocytes. To understand neutrophil migration from BM, we studied the vascular system of murine long bones. Here, in a mouse model, we show that hundreds of capillaries originate in BM, traverse cortical bone perpendicularly along the shaft and connect to the periosteal circulation. Structures similar to these trans-cortical vessels (TCVs) also exist in human limb bones. TCVs express arterial or venous markers and transport neutrophils. Furthermore, over 80% of arterial and 59% of venous blood passes through TCVs. Genetic and drug-mediated modulation of osteoclast count and activity leads to substantial changes in TCV numbers. In a murine model of chronic arthritic bone inflammation, new TCVs develop within weeks. Our data indicate that TCVs are a central component of the closed circulatory system in long bones and may represent an important route for immune cell export from BM.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.

Fig. 1: Identification of blood vessels in the shaft of murine long bones.
Fig. 2: Characterization and size verification of different vessel types by multiple imaging techniques.
Fig. 3: Characterization of TCVs and blood flow in murine tibiae.
Fig. 4: Trans-cortical canals are remodelled by osteoclasts.
Fig. 5: Chronic, but not acute, arthritis affects TCV formation.
Fig. 6: Evidence for trans-cortical blood flow in human long bones.

Data availability

The data that support the findings of this study are available from the corresponding author upon request


  1. 1.

    Faury, G. Function-structure relationship of elastic arteries in evolution: from microfibrils to elastin and elastic fibres. Pathol. Biol. (Paris) 49, 310–325 (2001).

    CAS  Google Scholar 

  2. 2.

    Bettex, D. A., Pretre, R. & Chassot, P. G. Is our heart a well-designed pump? The heart along animal evolution. Eur. Heart J. 35, 2322–2332 (2014).

    PubMed  Google Scholar 

  3. 3.

    Pittman, R. N. Oxygen transport in the microcirculation and its regulation. Microcirculation 20, 117–137 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  4. 4.

    Aitsebaomo, J., Portbury, A. L., Schisler, J. C. & Patterson, C. Brothers and sisters: molecular insights into arterial-venous heterogeneity. Circ. Res. 103, 929–939 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  5. 5.

    Sivaraj, K. K. & Adams, R. H. Blood vessel formation and function in bone. Development 143, 2706–2715 (2016).

    CAS  PubMed  Google Scholar 

  6. 6.

    Blumer, M. J., Longato, S. & Fritsch, H. Structure, formation and role of cartilage canals in the developing bone. Ann. Anat. 190, 305–315 (2008).

    PubMed  Google Scholar 

  7. 7.

    Sommerfeldt, D. W. & Rubin, C. T. Biology of bone and how it orchestrates the form and function of the skeleton. Eur. Spine J. 10(Suppl 2), S86–S95 (2001).

    PubMed  PubMed Central  Google Scholar 

  8. 8.

    Zimmermann, E. A., Busse, B. & Ritchie, R. O. The fracture mechanics of human bone: influence of disease and treatment. Bone Rep. 4, 743 (2015).

    Google Scholar 

  9. 9.

    Nair, A. K., Gautieri, A., Chang, S. W. & Buehler, M. J. Molecular mechanics of mineralized collagen fibrils in bone. Nat. Commun. 4, 1724 (2013).

    PubMed  PubMed Central  Google Scholar 

  10. 10.

    Simpson, A. H. The blood supply of the periosteum. J. Anat. 140(Pt 4), 697–704 (1985).

    PubMed  PubMed Central  Google Scholar 

  11. 11.

    Xie, Y. et al. Detection of functional haematopoietic stem cell niche using real-time imaging. Nature 457, 97–101 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  12. 12.

    Roche, B. et al. Structure and quantification of microvascularisation within mouse long bones: what and how should we measure? Bone 50, 390–399 (2012).

    PubMed  Google Scholar 

  13. 13.

    Acar, M. et al. Deep imaging of bone marrow shows non-dividing stem cells are mainly perisinusoidal. Nature 526, 126–130 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  14. 14.

    Chen, J. Y. et al. Hoxb5 marks long-term haematopoietic stem cells and reveals a homogenous perivascular niche. Nature 530, 223–227 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  15. 15.

    Köhler, A. et al. Altered cellular dynamics and endosteal location of aged early hematopoietic progenitor cells revealed by time-lapse intravital imaging in long bones. Blood 114, 290–298 (2009).

    PubMed  PubMed Central  Google Scholar 

  16. 16.

    Spencer, J. A. et al. Direct measurement of local oxygen concentration in the bone marrow of live animals. Nature 508, 269–273 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  17. 17.

    Köhler, A. et al. G-CSF mediated thrombopoietin release triggers neutrophil motility and mobilization from bone marrow via induction of Cxcr2 ligands. Blood 117, 4349–4357 (2011).

    PubMed  PubMed Central  Google Scholar 

  18. 18.

    Devi, S. et al. Neutrophil mobilization via plerixafor-mediated CXCR4 inhibition arises from lung demargination and blockade of neutrophil homing to the bone marrow. J. Exp. Med. 210, 2321–2336 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  19. 19.

    Junt, T. et al. Dynamic visualization of thrombopoiesis within bone marrow. Science 317, 1767–1770 (2007).

    CAS  PubMed  Google Scholar 

  20. 20.

    Chatfield-Ball, C., Boyle, P., Autier, P., van Wees, S. H. & Sullivan, R. Lessons learned from the casualties of war: battlefield medicine and its implication for global trauma care. J. R. Soc. Med. 108, 93–100 (2015).

    PubMed  PubMed Central  Google Scholar 

  21. 21.

    Clemency, B. et al. Intravenous vs. intraosseous access and return of spontaneous circulation during out of hospital cardiac arrest. Am. J. Emerg. Med. 35, 222–226 (2017).

    PubMed  Google Scholar 

  22. 22.

    Von Hoff, D. D., Kuhn, J. G., Burris, H. A. 3rd & Miller, L. J. Does intraosseous equal intravenous? A pharmacokinetic study. Am. J. Emerg. Med. 26, 31–38 (2008).

    Google Scholar 

  23. 23.

    Morelli, D., Menard, S., Cazzaniga, S., Colnaghi, M. I. & Balsari, A. Intratibial injection of an anti-doxorubicin monoclonal antibody prevents drug-induced myelotoxicity in mice. Br. J. Cancer 75, 656–659 (1997).

    CAS  PubMed  PubMed Central  Google Scholar 

  24. 24.

    Gunzer, M. Traps and hyper inflammation - new ways that neutrophils promote or hinder survival. Br. J. Haematol. 164, 188–199 (2014).

    Google Scholar 

  25. 25.

    Panopoulos, A. D. & Watowich, S. S. Granulocyte colony-stimulating factor: molecular mechanisms of action during steady state and ‘emergency’ hematopoiesis. Cytokine 42, 277–288 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  26. 26.

    Hasenberg, A. et al. Catchup: a mouse model for imaging-based tracking and modulation of neutrophil granulocytes. Nat. Methods 12, 445–452 (2015).

    CAS  PubMed  Google Scholar 

  27. 27.

    Herisson, F. et al. Direct vascular channels connect skull bone marrow and the brain surface enabling myeloid cell migration. Nat. Neurosci. 21, 1209–1217 (2018).

    CAS  PubMed  Google Scholar 

  28. 28.

    Morrison, S. J. & Scadden, D. T. The bone marrow niche for haematopoietic stem cells. Nature 505, 327–334 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  29. 29.

    Itkin, T. et al. Distinct bone marrow blood vessels differentially regulate haematopoiesis. Nature 532, 323–328 (2016).

    CAS  PubMed  Google Scholar 

  30. 30.

    Kusumbe, A. P., Ramasamy, S. K. & Adams, R. H. Coupling of angiogenesis and osteogenesis by a specific vessel subtype in bone. Nature 507, 323–328 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  31. 31.

    Ramasamy, S. K. et al. Regulation of hematopoiesis and osteogenesis by blood vessel-derivedsignals. Annu. Rev. Cell. Dev. Biol. 32, 649–675 (2016).

    CAS  PubMed  Google Scholar 

  32. 32.

    De Bruyn, P. P., Breen, P. C. & Thomas, T. B. The microcirculation of the bone marrow. Anat. Rec. 168, 55–68 (1970).

    PubMed  Google Scholar 

  33. 33.

    Klingberg, A. et al. Fully automated evaluation of total glomerular number and capillary tuft size in nephritic kidneys using lightsheet microscopy. J. Am. Soc. Nephrol. 28, 452–459 (2017).

    CAS  PubMed  Google Scholar 

  34. 34.

    Hanke, R., Fuchs, T. & Uhlmann, N. X-ray based methods for non-destructive testing and material characterization. Nucl. Instrum. Meth. A 591, 14–18 (2008).

    CAS  Google Scholar 

  35. 35.

    Stelzer, E. H. Light-sheet fluorescence microscopy for quantitative biology. Nat. Methods 12, 23–26 (2014).

    Google Scholar 

  36. 36.

    Schneider, P., Voide, R., Stampanoni, M., Donahue, L. R. & Muller, R. The importance of the intracortical canal network for murine bone mechanics. Bone 53, 120–128 (2013).

    PubMed  Google Scholar 

  37. 37.

    Muller, A. M. et al. Expression of the endothelial markers PECAM-1, vWf, and CD34 in vivo and in vitro. Exp. Mol. Pathol. 72, 221–229 (2002).

    PubMed  Google Scholar 

  38. 38.

    Chi, J. T. et al. Endothelial cell diversity revealed by global expression profiling. Proc. Natl Acad. Sci. USA 100, 10623–10628 (2003).

    CAS  PubMed  Google Scholar 

  39. 39.

    Williams, S. P. et al. Genome-wide functional analysis reveals central signaling regulators of lymphatic endothelial cell migration and remodeling. Sci Signal. 10, eaal2987 (2017).

    PubMed  Google Scholar 

  40. 40.

    Ramasamy, S. K. et al. Blood flow controls bone vascular function and osteogenesis. Nat. Commun. 7, 13601 (2016).

    PubMed  PubMed Central  Google Scholar 

  41. 41.

    Faust, N., Varas, F., Kelly, L. M., Heck, S. & Graf, T. Insertion of enhanced green fluorescent protein into the lysozyme gene creates mice with green fluorescent granulocytes and macrophages. Blood 96, 719–726 (2000).

    CAS  PubMed  Google Scholar 

  42. 42.

    Parfitt, A. M. Osteonal and hemi-osteonal remodeling: the spatial and temporal framework for signal traffic in adult human bone. J. Cell. Biochem. 55, 273–286 (1994).

    CAS  PubMed  Google Scholar 

  43. 43.

    Lassen, N. E. et al. Coupling of bone resorption and formation in real time: new knowledge gained from human Haversian BMUs. J. Bone Miner. Res. 32, 1395–1405 (2017).

    CAS  PubMed  Google Scholar 

  44. 44.

    Zebaze, R. M. et al. Differing effects of denosumab and alendronate on cortical and trabecular bone. Bone 59, 173–179 (2014).

    CAS  PubMed  Google Scholar 

  45. 45.

    Keffer, J. et al. Transgenic mice expressing human tumour necrosis factor: a predictive genetic model of arthritis. EMBO J. 10, 4025–4031 (1991).

    CAS  PubMed  PubMed Central  Google Scholar 

  46. 46.

    Houben, A. et al. beta-catenin activity in late hypertrophic chondrocytes locally orchestrates osteoblastogenesis and osteoclastogenesis. Development 143, 3826–3838 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  47. 47.

    Schubert, D., Maier, B., Morawietz, L., Krenn, V. & Kamradt, T. Immunization with glucose-6-phosphate isomerase induces T cell-dependent peripheral polyarthritis in genetically unaltered mice. J. Immunol. 172, 4503–4509 (2004).

    CAS  PubMed  Google Scholar 

  48. 48.

    Billiau, A. & Matthys, P. Modes of action of Freund’s adjuvants in experimental models of autoimmune diseases. J. Leukoc. Biol. 70, 849–860 (2001).

    CAS  PubMed  Google Scholar 

  49. 49.

    Frey, O. et al. Regulatory T cells control the transition from acute into chronic inflammation in glucose-6-phosphate isomerase-induced arthritis. Ann. Rheum. Dis. 69, 1511–1518 (2010).

    CAS  PubMed  Google Scholar 

  50. 50.

    Win, S. J., Kühl, A. A., Sparwasser, T., Hünig, T. & Kamradt, T. In vivo activation of Treg cells with a CD28 superagonist prevents and ameliorates chronic destructive arthritis in mice. Eur. J. Immunol. 46, 1193–1202 (2016).

    CAS  PubMed  Google Scholar 

  51. 51.

    Wehmeyer, C. et al. Sclerostin inhibition promotes TNF-dependent inflammatory joint destruction. Sci. Transl. Med. 8, 330ra335 (2016).

    Google Scholar 

  52. 52.

    Irmler, I. M. et al. 18 F-Fluoride positron emission tomography/computed tomography for noninvasive in vivo quantification of pathophysiological bone metabolism in experimental murine arthritis. Arthritis Res. Ther. 16, R155 (2014).

    PubMed  PubMed Central  Google Scholar 

  53. 53.

    Pastille, E. et al. Transient ablation of regulatory T cells improves antitumor immunity in colitis-associated colon cancer. Cancer Res. 74, 4258–4269 (2014).

    CAS  PubMed  Google Scholar 

  54. 54.

    Gunal, I., Ozcelik, A., Gokturk, E., Ada, S. & Demirtas, M. Correlation of magnetic resonance imaging and intraoperative punctate bleeding to assess the vascularity of scaphoid nonunion. Arch. Orthop. Trauma. Surg. 119, 285–287 (1999).

    CAS  PubMed  Google Scholar 

  55. 55.

    Voide, R. et al. The importance of murine cortical bone microstructure for microcrack initiation and propagation. Bone 49, 1186–1193 (2011).

    CAS  PubMed  Google Scholar 

  56. 56.

    Schneider, P. et al. Ultrastructural properties in cortical bone vary greatly in two inbred strains of mice as assessed by synchrotron light based micro- and nano-CT. J. Bone Miner. Res. 22, 1557–1570 (2007).

    PubMed  Google Scholar 

  57. 57.

    Mercier, F. E., Ragu, C. & Scadden, D. T. The bone marrow at the crossroads of blood and immunity. Nat. Rev. Immunol. 12, 49–60 (2011).

    PubMed  PubMed Central  Google Scholar 

  58. 58.

    Lai, X. et al. The dependences of osteocyte network on bone compartment, age, and disease. Bone Res. 3, 15009 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  59. 59.

    Hasegawa, T. et al. Three-dimensional ultrastructure of osteocytes assessed by focused ion beam-scanning electron microscopy (FIB-SEM). Histochem. Cell. Biol. 149, 423–432 (2018).

    CAS  PubMed  Google Scholar 

  60. 60.

    Smit, T. H., Burger, E. H. & Huyghe, J. M. A case for strain-induced fluid flow as a regulator of BMU-coupling and osteonal alignment. J. Bone Miner. Res. 17, 2021–2029 (2002).

    PubMed  Google Scholar 

  61. 61.

    Delgado-Calle, J. et al. MMP14 is a novel target of PTH signaling in osteocytes that controls resorption by regulating soluble RANKL production. FASEB J. 32, 2878–2890 (2018).

    PubMed  Google Scholar 

  62. 62.

    Bellido, T. Osteocyte-driven bone remodeling. Calcif. Tissue Int. 94, 25–34 (2014).

    CAS  PubMed  Google Scholar 

  63. 63.

    Schaffler, M. B., Cheung, W. Y., Majeska, R. & Kennedy, O. Osteocytes: master orchestrators of bone. Calcif. Tissue Int. 94, 5–24 (2014).

    CAS  PubMed  Google Scholar 

  64. 64.

    Honma, M. et al. RANKL subcellular trafficking and regulatory mechanisms in osteocytes. J. Bone Miner. Res. 28, 1936–1949 (2013).

    CAS  PubMed  Google Scholar 

  65. 65.

    Chen, K., Pittman, R. N. & Popel, A. S. Nitric oxide in the vasculature: where does it come from and where does it go? A quantitative perspective. Antioxid. Redox Signal. 10, 1185–1198 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  66. 66.

    Huang, N. F., Fleissner, F., Sun, J. & Cooke, J. P. Role of nitric oxide signaling in endothelial differentiation of embryonic stem cells. Stem. Cells Dev. 19, 1617–1626 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  67. 67.

    Morbidelli, L., Donnini, S. & Ziche, M. Role of nitric oxide in the modulation of angiogenesis. Curr. Pharm. Des. 9, 521–530 (2003).

    CAS  PubMed  Google Scholar 

  68. 68.

    Birukova, A. A. et al. Prostaglandins PGE2 and PGI2 promote endothelial barrier. Exp. Cell Res. 313, 2504–2520 (2007).

    CAS  PubMed  PubMed Central  Google Scholar 

  69. 69.

    Pai, R. et al. PGE(2) stimulates VEGF expression in endothelial cells via ERK2/JNK1 signaling pathways. Biochem. Biophys. Res. Commun. 286, 923–928 (2001).

    CAS  PubMed  Google Scholar 

  70. 70.

    Werner, D. et al. Early changes of the cortical micro-channel system in the bare area of the joints of patients with rheumatoid arthritis. Arthritis Rheumatol. 69, 1580–1587 (2017).

    PubMed  Google Scholar 

  71. 71.

    Maggiano, I. S. et al. Three-dimensional reconstruction of Haversian systems in human cortical bone using synchrotron radiation-based micro-CT: morphology and quantification of branching and transverse connections across age. J. Anat. 228, 719–732 (2016).

    PubMed  PubMed Central  Google Scholar 

  72. 72.

    Suen, P. K. & Qin, L. Sclerostin, an emerging therapeutic target for treating osteoporosis and osteoporotic fracture: a general review. J. Orthop. Transl. 4, 1–13 (2016).

    Google Scholar 

  73. 73.

    Tomlinson, R. E. & Silva, M. J. Skeletal blood flow in bone repair and maintenance. Bone Res. 1, 311–322 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  74. 74.

    Green, D. E. & Rubin, C. T. Consequences of irradiation on bone and marrow phenotypes, and its relation to disruption of hematopoietic precursors. Bone 63, 87–94 (2014).

    CAS  PubMed  Google Scholar 

  75. 75.

    Wright, L. E. et al. Single-limb irradiation induces local and systemic bone loss in a murine model. J. Bone Miner. Res. 30, 1268–1279 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  76. 76.

    Yang, B. et al. Effect of radiation on the expression of osteoclast marker genes in RAW264.7 cells. Mol. Med. Rep. 5, 955–958 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  77. 77.

    Zhang, J. et al. Differences in responses to X-ray exposure between osteoclast and osteoblast cells. J. Radiat. Res. 58, 791–802 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  78. 78.

    Brault, V. et al. Inactivation of the β-catenin gene by Wnt1-Cre-mediated deletion results in dramatic brain malformation and failure of craniofacial development. Development 128, 1253–1264 (2001).

    CAS  PubMed  PubMed Central  Google Scholar 

  79. 79.

    Gebhard, S. et al. Specific expression of Cre recombinase in hypertrophic cartilage under the control of a BAC-Col10a1 promoter. Matrix Biol.: J. Int. Soc. Matrix Biol. 27, 693–699 (2008).

    CAS  Google Scholar 

  80. 80.

    Harada, N. et al. Intestinal polyposis in mice with a dominant stable mutation of the beta-catenin gene. EMBO J. V. 18, 5931–5942 (1999).

    CAS  Google Scholar 

  81. 81.

    Wan, Y. Y. & Flavell, R. A. Identifying Foxp3-expressing suppressor T cells with a bicistronic reporter. Proc. Natl Acad. Sci. USA 102, 5126–5131 (2005).

    CAS  PubMed  Google Scholar 

  82. 82.

    Lahl, K. et al. Selective depletion of Foxp3+regulatory T cells induces a scurfy-like disease. J. Exp. Med. 204, 57–63 (2007).

    CAS  PubMed  PubMed Central  Google Scholar 

  83. 83.

    Madisen, L. et al. A robust and high-throughput Cre reporting and characterization system for the whole mouse brain. Nat. Neurosci. 13, 133–140 (2010).

    CAS  PubMed  Google Scholar 

  84. 84.

    Shearing, P. R. & Brandon, N. P. X-ray nano computerised tomography of SOFC electrodes using a focused ion beam sample-preparation technique. J. Eur. Ceram. Soc. 30, 1809–1814 (2010).

    CAS  Google Scholar 

  85. 85.

    Kraff, O. et al. An eight-channel phased array RF coil for spine MR imaging at 7 T. Invest. Radiol. 44, 734–740 (2009).

    PubMed  Google Scholar 

  86. 86.

    Rietsch, S. H. G. et al. An 8-channel transceiver 7-channel receive RF coil setup for high SNR ultrahigh-field MRI of the shoulder at 7T. Med. Phys. 44, 6195–6208 (2017).

    PubMed  Google Scholar 

  87. 87.

    Jiru, F. & Klose, U. Fast 3D radiofrequency field mapping using echo-planar imaging. Magn. Reson. Med. 56, 1375–1379 (2006).

    CAS  PubMed  Google Scholar 

  88. 88.

    Johst, S. et al. Time-of-flight magnetic resonance angiography at 7 T using venous saturation pulses with reduced flip angles. Invest. Radiol. 47, 445–450 (2012).

    PubMed  Google Scholar 

  89. 89.

    Griswold, M. A. et al. Generalized autocalibrating partially parallel acquisitions (GRAPPA). Magn. Reson. Med. 47, 1202–1210 (2002).

    Google Scholar 

Download references


We thank the IMaging Center ESsen (IMCES: Light Microscopy Unit (LMU), the IMCES Electron Microscopy Unit (EMU) and the Optical Imaging Centre Erlangen (OICE: for support with imaging. In addition, we wish to thank R. Burgemeister (Carl Zeiss Microscopy) for support through the Zeiss labs@location program and M. Löffler (DCN, TU Dresden) for his help with X-ray microscopy. J. Kamradt is acknowledged for critical reading of the manuscript. This work was supported by funds from the German Research Foundation (SPP1480 Immunobone ) to M.G., G.S., T.K., A.I.G., A.V., G.K. and M.H.; FZT 111 (Center for Regenerative Therapies Dresden, Cluster of Excellence) to A.I.G.; the Collaborative Research Centre (CRC) 1181 to G.K., M.H. and G.S.; the German Ministry of Education and Research (BMBF NeuroImpa 01EC1403A) to T.K.; and the European Union (EU HEALTH-2013-INNOVATION-1, MATHIAS) to M.G.. The work of G.S. was also supported by the Innovative Medicine Initiative (IMI)-funded project RTCure and the European Research Council (ERC) Synergy grant NanoScope.

Author information




A.K., I.H., D.W., S.C., S.M., L.B., A.B., S.M., S.H., K.Z., S.L., W.B., A.O., R.D., J.V.S., A.I.G., A.A., M.W. and A.H. performed all optical imaging and animal and wet-lab experiments. O.K. and H.H.Q. performed 7 T magnetic resonance imaging measurements. K.G., M.J., S.L. and M.D. performed surgical procedures on human patients. M.R., M.H. and S.V. performed SEM imaging. L.K., S.C. and M.H. performed XRM imaging. D.H. developed the algorithm for and analysed blood flow images. M.G. conceived of and supervised the study and wrote the manuscript with the help of A.K, A.M.W., D.R.E., A.V., G.K., T.K., G.S. and A.H. All authors contributed to discussions and writing of the manuscript.

Corresponding authors

Correspondence to Anja Hasenberg or Matthias Gunzer.

Ethics declarations

Competing interests

The authors declare no competing interests.

Additional information

Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary information

Supplementary Information

Supplementary Figures 1–9 and Supplementary Tables 1–5

Reporting Summary

Supplementary Video 1

X-ray microscopy (XRM) imaging of a C57BL/6J tibia. X-ray microscopy allows visualization of the compact bone structures of a C57BL/6J tibia, including multiple pores on the bone surface. Optical sectioning of the 3D reconstruction identifies bone pores as canals, traversing the entire compact bone. Zooming into the 3D reconstruction allows the differentiation of bone canals from osteocyte lacunae in the compact bone. Experiments were performed three times individually, with similar results

Supplementary Video 2

XRM imaging of C57BL/6J bone canals. Multiple pores can be detected on the surface of a tibia via XRM. While one dominant pore is observed in the posterior metaphyseal bone, numerous smaller pores can be observed all over the bone surface. These pores form canals traversing the entire compact bone. This allows a connection between the bone surface and bone marrow lacuna, shown via an optical journeythrough one exemplary canal. Experiments were performed three times individually, with similar results

Supplementary Video 3

LSFM of a simpleClear-treated C57BL/6J tibia. LSFM of a simpleClear optically cleared tibia allows visualization of the entire bone. While there are multiple blood vessels (CD31, red) detected on the bone surface (autofluorescence, grey) forming the periosteum, one dominant vessel is found at the posterior diaphysis. This vessel is entering the bone shaft and ramifies in the bone marrow. Additionally, a central vessel canal can be observed in the centre of the bone marrow, as well as multiple trans-cortical vessels connecting the bone marrow with the bone surface by traversing the compact bone. Experiments were performed six times individually, with similar results

Supplementary Video 4

LSFM visualization of a nutrient artery and nutrient sinus. Double staining of CD31 and SCA-1 allows the identification of veins (CD31+SCA-1, blue) and arteries (CD31+SCA-1+, red) in a simpleClear-treated C57BL/6J tibia. The dominant vessel at the posterior diaphysis shown in Supplementary Video 3 is thereby identified as a nutrient artery, which ramifies in the marrow. The central sinus is exiting the bone shaft (autofluorescence, grey) at the anterior diaphysis. Experiments were performed 15 times individually, with similar results

Supplementary Video 5

The periosteal vessel network of a C57BL/6J tibia. High-magnification TPLSM of a simpleClear-treated tibia allows visualization of the arterial (CD31+SCA-1+, red) and venous (CD31+SCA-1, blue) vessel network in the tissue surrounding the compact bone (SHG, grey). According to their structural orientation, the muscle vascularization can be distinguished from the periosteum, forming a dense vessel network along the bone surface. Experiments were performed five times individually, with similar results

Supplementary Video 6

Visualization of arterial and venous connections in a C57BL/6J fibula. High-magnification TPLSM of a fibula showing venous (CD31+SCA-1, blue) and arterial (CD31+SCA-1+) vessels within the simpleClear-treated bone (SHG, grey). Multiple venous and arterial TCVs are connecting the periosteum to the marrow vascularization within the fibula. In the marrow, the venous central sinus is connected to the sinusoidal network. Additionally, arteries are running along the bone shaft and connected to the sinusoids. Experiments were performed five times individually, with similar results

Supplementary Video 7

Blood flow in different cortical vessels. The blood flow in different types of cortical vessel is visualized by intra-vital TPLSM of LysM-EGFP tibiae (SHG, grey). TCVs, nutrient arteries (NAs) and the bone-exiting sinus differ not only in diameter but also in speed of blood flow (rhodamine dextran, red) and cell transport (EGFP, green) (n = 25 TCV, 7 NA and 5 central sinus scans)

Supplementary Video 8

Intra-vital imaging of G-CSF mobilization in a LysM-EGFP mouse. Intra-vital imaging of the tibial surface (SHG, grey) shows blood flow (rhodamine dextran, red), but only rarely cell transport (EGFP, green), through TCVs under untreated conditions. About 20 min after application of G-CSF, an increase in cell transport by the bloodstream, as well as active cell migration against the direction of blood flow, can be observed in TCVs and a nutrient artery. Experiments were performed five times individually, with similar results

Supplementary Video 9

Location of osteoclasts at the endosteum and in trans-cortical canals. LSFM imaging of a simpleClear-treated CX3CR1-cre;tdTomato tibia shows high numbers of osteoclasts (red) located along the endosteum of the diaphysis. Furthermore, osteoclasts can be found in the vascularized (CD31, turquoise) trans-cortical canals (TCCs) preferentially located in the centre of the compact bone (autofluorescence, grey). Widening of TCCs at these locations may indicate bone remodelling and formation of new TCVs emanating from existing TCCs. Experiments were performed eight times individually, with similar results

Supplementary Video 10

Osteoclasts remodelling trans-cortical canals. The generation of SHG signals (grey) via TPLSM imaging enables the visualization of compact bone tissue. The widened TCC area associated with osteoclast location (CX3CR1-cre;tdTomato, red) lacks SHG signals, suggesting the formation of a resorption lacuna where the compact bone tissue is dissolved by the adjacent osteoclast. Experiments were performed three times individually, with similar results

Supplementary Video 11

7T MRI imaging of a human shank. 3D reconstruction of 7T MRT data allows visualization of a human shank and identification of specific structures including muscle tissue (brown), compact bone (grey), arteries (red) and veins (blue). Optical clipping of the tibia shows a nutrient artery entering the bone shaft and a central sinus running parallel in the bone cavity. Experiments were performed twice individually, with similar results

Supplementary Video 12

Blood flow egression from human bone. Surgical exposure of the human femoral neck shows blood egression from multiple pores on the bone surface. Experiments were performed twice individually, with similar results

Supplementary Video 13

Whole-mount-stained and simpleCLEAR-cleared human femoral neck. Whole-mount staining of a human femoral neck with CD31 (turquoise) and α-smooth muscle actin (α-SMA, red) permits visualization of arterial and venous vessels in the human tissue sample. A large artery entering the compact bone (autofluorescence, grey) from the periosteum and a small artery traversing trabeculae in the bone marrow can be observed. Experiments were performed three times individually, with similar results

Supplementary Video 14

TCVs in the human femoral neck. Staining of veins (CD31+SCA-1, turquoise) and arteries (CD31+SCA-1+, red) in the human femoral neck enables visualization not only of the Haversian vascular system in compact bone (autofluorescence, grey), but also of the presence of dTCVs directly connecting the bone marrow with the periosteal vessel network. Experiments were performed four times individually, with similar results

Supplementary Video 15

Osteoclast–osteocyte interaction in TCVs. 3D rendering of a confocal scanned histological bone section shows an osteocyte in the compact bone, identified by its characteristic dendritic-like morphology (phalloidin, green, DAPI blue). The osteocyte dendrites are connected to an osteoclast (Cx3cr1-cre;tdTomato, red) located in a TCV (phalloidin, green). Experiments were performed six times individually, with similar results

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Grüneboom, A., Hawwari, I., Weidner, D. et al. A network of trans-cortical capillaries as mainstay for blood circulation in long bones. Nat Metab 1, 236–250 (2019).

Download citation

Further reading


Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing