Skip to main content

Thank you for visiting nature.com. 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.

  • Article
  • Published:

Nano-analytical electron microscopy reveals fundamental insights into human cardiovascular tissue calcification

Nature Materials volume 12, pages 576–583 (2013)Cite this article

Subjects

This article has been updated

Abstract

The accumulation of calcified material in cardiovascular tissue is thought to involve cytochemical, extracellular matrix and systemic signals; however, its precise composition and nanoscale architecture remain largely unexplored. Using nano-analytical electron microscopy techniques, we examined valves, aortae and coronary arteries from patients with and without calcific cardiovascular disease and detected spherical calcium phosphate particles, regardless of the presence of calcific lesions. We also examined lesions after sectioning with a focused ion beam and found that the spherical particles are composed of highly crystalline hydroxyapatite that crystallographically and structurally differs from bone mineral. Taken together, these data suggest that mineralized spherical particles may play a fundamental role in calcific lesion formation. Their ubiquitous presence in varied cardiovascular tissues and from patients with a spectrum of diseases further suggests that lesion formation may follow a common process. Indeed, applying materials science techniques to ectopic and orthotopic calcification has great potential to lend critical insights into pathophysiological processes underlying calcific cardiovascular disease.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: DDC-SEM micrographs of human aortic valve tissue.
Figure 2: Elemental analyses of dense structures identified on aortic valves.
Figure 3: Histograms describing the prevalence of dense calcium- and phosphorus-containing structures on the surface of aortic valves.
Figure 4: TEM images, EDS mapping and SAED analyses of aortic valve tissues sectioned with a FIB.
Figure 5: DDC-SEM micrographs of mitral valve, aorta and coronary artery, and TEM, SAED and EDS mapping of calcific lesions on coronary artery tissue sectioned using a FIB.

Similar content being viewed by others

Change history

  • 24 April 2013

    In the version of this Article originally published online, the first name of the fourth author was incorrect: it should have read 'Adrian H. Chester'. This error has been corrected in all versions of the Article.

References

  1. Butler, D. UN targets top killers. Nature 477, 260–261 (2011).

    Article  CAS  Google Scholar 

  2. Rajamannan, N. M. et al. Human aortic valve calcification is associated with an osteoblast phenotype. Circulation 107, 2181–2184 (2003).

    Article  Google Scholar 

  3. Lindroos, M., Kupari, M., Heikkila, J. & Tilvis, R. Prevalence of aortic valve abnormalities in the elderly: An echocardiographic study of a random population sample. J. Am. Coll. Cardiol. 21, 1220–1225 (1993).

    Article  CAS  Google Scholar 

  4. Nkomo, V. T. et al. Burden of valvular heart diseases: A population-based study. Lancet 368, 1005–1011 (2006).

    Article  Google Scholar 

  5. Roger, V. L. et al. Heart disease and stroke statistics—2011 update. Circulation 123, e18–e209 (2011).

    Article  Google Scholar 

  6. Iung, B. & Vahanian, A. Epidemiology of valvular heart disease in the adult. Nature Rev. Cardiol. 8, 162–172 (2011).

    Article  Google Scholar 

  7. Sekikawa, A. et al. Aortic stiffness and calcification in men in a population-based international study. Atherosclerosis 222, 473–477 (2012).

    Article  CAS  Google Scholar 

  8. Sage, A. P., Tintut, Y. & Demer, L. L. Regulatory mechanisms in vascular calcification. Nature Rev. Cardiol. 7, 528–536 (2010).

    Article  CAS  Google Scholar 

  9. Langanay, T. et al. Aortic valve replacement in the elderly: The real life. Ann. Thorac. Surg. 93, 70–78 (2012).

    Article  Google Scholar 

  10. Thomas, M. et al. Thirty-day results of the sapien aortic bioprosthesis european outcome (source) registry. Circulation 122, 62–69 (2010).

    Article  Google Scholar 

  11. Duer, M. J. et al. Mineral surface in calcified plaque is like that of bone further evidence for regulated mineralization. Arterioscler. Thromb. Vasc. Biol. 28, 2030–2034 (2008).

    Article  CAS  Google Scholar 

  12. Mody, N., Tintut, Y., Radcliff, K. & Demer, L. Vascular calcification and its relation to bone calcification: Possible underlying mechanisms. J. Nucl. Cardiol. 10, 177–183 (2003).

    Article  Google Scholar 

  13. Delogne, C., Lawford, P. V., Habesch, S. M. & Carolan, V. A. Characterization of the calcification of cardiac valve bioprostheses by environmental scanning electron microscopy and vibrational spectroscopy. J. Microsc. 228, 62–77 (2007).

    Article  CAS  Google Scholar 

  14. Weska, R. F. et al. Natural and prosthetic heart valve calcification: Morphology and chemical composition characterization. Artif. Organs 34, 311–318 (2010).

    Article  CAS  Google Scholar 

  15. Demer, L. L. & Tintut, Y. Vascular calcification: Pathobiology of a multifaceted disease. Circulation 117, 2938–2948 (2008).

    Article  Google Scholar 

  16. Fratzl, P. & Weinkamer, R. Nature’s hierarchical materials. Prog. Mater. Sci. 52, 1263–1334 (2007).

    Article  CAS  Google Scholar 

  17. Gentleman, E. et al. Comparative materials differences revealed in engineered bone as a function of cell-specific differentiation. Nature Mater. 8, 763–770 (2009).

    Article  CAS  Google Scholar 

  18. Mahamid, J. et al. Mapping amorphous calcium phosphate transformation into crystalline mineral from the cell to the bone in zebrafish fin rays. Proc. Natl Acad. Sci. USA 107, 6316–6321 (2010).

    Article  CAS  Google Scholar 

  19. Biltz, R. M. & Pellegri, E. D. Chemical anatomy of bone I. A comparative study of bone composition in 16 vertebrates. J. Bone Joint Surg.-Am. A 51, 456–466 (1969).

    Article  CAS  Google Scholar 

  20. Mohler, E. R. III et al. Bone formation and inflammation in cardiac valves. Circulation 103, 1522–1528 (2001).

    Article  Google Scholar 

  21. Rajamannan, N. M. et al. Calcific aortic valve disease: Not simply a degenerative process. Circulation 124, 1783–1791 (2011).

    Article  Google Scholar 

  22. Su, X., Sun, K., Cui, F. Z. & Landis, W. J. Organization of apatite crystals in human woven bone. Bone 32, 150–162 (2003).

    Article  CAS  Google Scholar 

  23. Mohr, W. & Görz, E. Verkalkungen der temporalarterien - ihre morphogenese im vergleich zur physiologischen osteogenese. Z. Kardiol. 92, 60–72 (2003).

    Article  CAS  Google Scholar 

  24. Mohr, W. & Gorz, E. Does calcification of arteriosclerotic vessels imitate osteogenesis? Pathomorphological study of arteriosclerotic plaques. Z. Kardiol. 91, 212–232 (2002).

    Article  CAS  Google Scholar 

  25. Kockx, M. M., Muhring, J., Bortier, H., DeMeyer, G. R. Y. & Jacob, W. Biotin- or digoxigenin-conjugated nucleotides bind to matrix vesicles in atherosclerotic plaques. Am. J. Pathol. 148, 1771–1777 (1996).

    CAS  Google Scholar 

  26. Johnston, K. W. et al. Determining the quantity and character of carotid artery embolic debris by electron microscopy and energy dispersive spectroscopy—discussion. J. Vasc. Surg. 45, 724–725 (2007).

    Article  Google Scholar 

  27. Miller, V. M. et al. Evidence of nanobacterial-like structures in calcified human arteries and cardiac valves. Am. J. Physiol. Heart Circ. Physiol. 287, H1115–H1124 (2004).

    Article  CAS  Google Scholar 

  28. Koutsopoulos, S., Kontogeorgou, A., Dalas, E. & Petroheilos, J. Calcification of porcine and human cardiac valves: testing of various inhibitors for antimineralization. J. Mater. Sci.-Mater. Med. 9, 421–424 (1998).

    Article  CAS  Google Scholar 

  29. Anderson, H. Matrix vesicles and calcification. Curr. Rheumatol. Rep. 5, 222–226 (2003).

    Article  Google Scholar 

  30. Boonrungsiman, S. et al. The role of intracellular calcium phosphate in osteoblast-mediated bone apatite formation. Proc. Natl Acad. Sci. USA 109, 14170–14175 (2012).

    Article  CAS  Google Scholar 

  31. Tanimura, A., McGregor, D. H. & Anderson, H. C. Matrix vesicles in artherosclerotic calcification. Proc. Soc. Exp. Biol. Med. 172, 173–177 (1983).

    Article  CAS  Google Scholar 

  32. Komori, T. et al. Targeted disruption of cbfa1 results in a complete lack of bone formation owing to maturational arrest of osteoblasts. Cell 89, 755–764 (1997).

    Article  CAS  Google Scholar 

  33. Satokata, I. et al. Msx2 deficiency in mice causes pleiotropic defects in bone growth and ectodermal organ formation. Nature Genet. 24, 391–395 (2000).

    Article  CAS  Google Scholar 

  34. Nakashima, K. et al. The novel zinc finger-containing transcription factor osterix is required for osteoblast differentiation and bone formation. Cell 108, 17–29 (2002).

    Article  CAS  Google Scholar 

  35. Jantou, V., Turmaine, M., West, G. D., Horton, M. A. & McComb, D. W. Focused ion beam milling and ultramicrotomy of mineralised ivory dentine for analytical transmission electron microscopy. Micron 40, 495–501 (2009).

    Article  CAS  Google Scholar 

  36. McNally, E. A., Schwarcz, H. P., Botton, G. A. & Arsenault, A. L. A model for the ultrastructure of bone based on electron microscopy of ion-milled sections. PLoS ONE 7, e29258 (2012).

    Article  CAS  Google Scholar 

  37. Landis, W. J., Hodgens, K. J., Arena, J., Song, M. J. & McEwen, B. F. Structural relations between collagen and mineral in bone as determined by high voltage electron microscopic tomography. Microsc. Res. Tech. 33, 192–202 (1996).

    Article  CAS  Google Scholar 

  38. Nudelman, F. et al. The role of collagen in bone apatite formation in the presence of hydroxyapatite nucleation inhibitors. Nature Mater. 9, 1004–1009 (2010).

    Article  CAS  Google Scholar 

  39. New, S. E. P. & Aikawa, E. Cardiovascular calcification: An inflammatory disease. Circ. J. 75, 1305–1313 (2011).

    Article  CAS  Google Scholar 

  40. Yuan, H. et al. Osteoinductive ceramics as a synthetic alternative to autologous bone grafting. Proc. Natl Acad. Sci. USA 107, 13614–13619 (2010).

    Article  CAS  Google Scholar 

  41. Yamasaki, H. & Sakai, H. Osteogenic response to porous hydroxyapatite ceramics under the skin of dogs. Biomaterials 13, 308–312 (1992).

    Article  CAS  Google Scholar 

  42. Yang, Z. J. et al. Osteogenesis in extraskeletally implanted porous calcium phosphate ceramics: Variability among different kinds of animals. Biomaterials 17, 2131–2137 (1996).

    Article  CAS  Google Scholar 

  43. Kurashina, K., Kurita, H., Wu, Q., Ohtsuka, A. & Kobayashi, H. Ectopic osteogenesis with biphasic ceramics of hydroxyapatite and tricalcium phosphate in rabbits. Biomaterials 23, 407–412 (2002).

    Article  CAS  Google Scholar 

  44. Le Nihouannen, D. et al. Ectopic bone formation by microporous calcium phosphate ceramic particles in sheep muscles. Bone 36, 1086–1093 (2005).

    Article  CAS  Google Scholar 

  45. Ducheyne, P. & Qiu, Q. Bioactive ceramics: the effect of surface reactivity on bone formation and bone cell function. Biomaterials 20, 2287–2303 (1999).

    Article  CAS  Google Scholar 

  46. LeGeros, R. Z. Properties of osteoconductive biomaterials: Calcium phosphates. Clin. Orthop. Rel. Res. 395, 81–98 (2002).

    Article  Google Scholar 

  47. LeGeros, R. Z. Calcium phosphate-based osteoinductive materials. Chem. Rev. 108, 4742–4753 (2008).

    Article  Google Scholar 

  48. Miller, J. D., Weiss, R. M. & Heistad, D. D. Calcific aortic valve stenosis: methods, models, and mechanisms. Circ. Res. 108, 1392–1412 (2011).

    Article  CAS  Google Scholar 

  49. Chen, J. H. & Simmons, C. A. Cell-matrix interactions in the pathobiology of calcific aortic valve disease critical roles for matricellular, matricrine, and matrix mechanics cues. Circ. Res. 108, 1510–1524 (2011).

    Article  CAS  Google Scholar 

  50. Engler, A. J., Sen, S., Sweeney, H. L. & Discher, D. E. Matrix elasticity directs stem cell lineage specification. Cell 126, 677–689 (2006).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

S.B. was supported by the Rosetrees Trust and the Junior Research Fellowship scheme at Imperial College London. E.G. was supported by a fellowship from the Wellcome Trust. K.L.C. was supported by a studentship from the British Heart Foundation Centre of Research Excellence at Imperial College London. M.M.S. gratefully acknowledges financial support from the Rosetrees Trust. We would like to thank K. Nitiputri for providing the murine osteoblast cells line MC3T3-E1. We would like to acknowledge the provision of preliminary animal tissues by W. Jahnen-Dechent and A. Kinkeldey.

Author information

Authors and Affiliations

  1. Department of Materials, Imperial College London, London SW7 2AZ, UK

    Sergio Bertazzo, Eileen Gentleman, Kristy L. Cloyd & Molly M. Stevens

  2. Institute of Biomedical Engineering, Imperial College London, London SW7 2AZ, UK

    Sergio Bertazzo, Eileen Gentleman, Kristy L. Cloyd & Molly M. Stevens

  3. Department of Craniofacial Development and Stem Cell Biology, King’s College London, London SE1 9RT, UK

    Eileen Gentleman

  4. National Heart and Lung Institute, Harefield Heart Science Centre, Imperial College London, Harefield, Middlesex UB9 6JH, UK

    Adrian H. Chester & Magdi H. Yacoub

  5. Department of Bioengineering, Imperial College London, London SW7 2AZ, UK

    Molly M. Stevens

Authors
  1. Sergio Bertazzo
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Eileen Gentleman
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Kristy L. Cloyd
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Adrian H. Chester
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Magdi H. Yacoub
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Molly M. Stevens
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

S.B. designed the study, performed sample preparation, conducted all the experimental work, carried out data interpretation and aided with manuscript preparation and writing. E.G. aided with study design and data interpretation and wrote most of the manuscript. K.L.C. helped with sample preparation. A.H.C. and M.H.Y helped with sample procurement and aided in data interpretation. M.M.S. supervised the study, aided with study design and data interpretation and revised the manuscript.

Corresponding author

Correspondence to Molly M. Stevens.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Information

Supplementary Information (PDF 1716 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Bertazzo, S., Gentleman, E., Cloyd, K. et al. Nano-analytical electron microscopy reveals fundamental insights into human cardiovascular tissue calcification. Nature Mater 12, 576–583 (2013). https://doi.org/10.1038/nmat3627

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nmat3627

This article is cited by

Search

Advanced search

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