Tag Archives: Kleoniki Lyroudia

Computerized Reconstruction of Pulpal Blood Vessels Examined under Confocal Microscope

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1 / Kleoniki Lyroudia2 / Lucie Kubinova3 / Georgia Karayannopoulou4 / Ioannis Marras5 / Ioannis Pitas5

1Private Dentist, Thessaloniki, Greece
2Aristotle University of Thessaloniki, Department of Endodontology, Dental School, Thessaloniki, Greece
3Academy of Sciences of the Czech Republic, Institute of Physiology, Department of Biomathematics, Prague, Czech Republic
4Aristotle University of Thessaloniki, Medical School, Thessaloniki, Greece
5Aristotle University of Thessaloniki, Department of Informatics, Thessaloniki, Greece


The purpose of this study was the evaluation of 3 different histological methods for studying pulpal blood vessels in combination with 2 types of confocal microscope and computer assisted 3-dimensional reconstruction. 10 human, healthy, free of restorations or caries teeth that were extracted for orthodontic reasons were used. From these teeth, the pulp tissues of 5 were removed, fixed in formalin solution, dehydrated and embedded in paraffin. Serial cross sections 5μm thick were taken from 3 of the above mentioned pulpal tissues and stained with CD34 according to the immunohistochemical ABC technique, while the rest 2 were stained with CD34 and Cy5 by means of immunofluorescence after serial cross sectioning of 10μm. 5 of the 10 teeth were fixed, decalcified, serial cross sectioned (30μm thickness) and stained with eosin. The physical sections were examined under 2 types of confocal laser microscope. Serial images were taken for each section, alignment of the images was followed and finally 3-dimensional reconstructions of the pulpal vessels were achieved.

The combined use of immunofluorescence, confocal microscope and automatic segmentation proved to be a useful method for the detailed study of pulpal vasculature. The above method provides deep knowledge of the form and spatial relationship even of the smallest pulpal blood vessels with neighbouring structures like odontoblasts, which are essential for the fully understanding of their role and function within the dental pulp.

Keywords: CD34; Cy5; Immunofluorescence; 3D reconstruction


  1. Avery J, Chiego D. Essentials of Oral Histology and Embryology. A Clinical Approach. Mosby Elsevier. 4thEdition. 2014.
  2. Luuko K, Kettunen P, Fristad I, Bergreen E. Structure and Functions of the dentin-pulp complex in Cohen’s Pathways of the pulp. Mosby Elsevier. 10thEdition. 2011.
  3. Vermeulen PB, Gasparini G, Fox SB, et al. Second international consensus on the methodology and criteria of evaluation of angiogenesis quantification in solid human tumors.Eur J Cancer, 2002; 38:1564-1579. [PubMed] [Crossref]
  4. Hasan J, Byers R, Jayson GC. Intra-tumoural microvessel density in human solid tumours.Br J Cancer, 2002; 86:1566-1577. [PubMed] [Crossref]
  5. Digka A, Lyroudia K, Jirasek T, Kasampalidis I, Karayannopoulou G, Kubinova L. Visualisation of human dental pulp vasculature by immunohistochemical and immunofluorescent detection of CD34: A comparative study.Aust Endod J, 2006; 32(3):101-106. [Crossref]
  6. Takahashi K, Kishi Y, Kim S. A scanning electron microscope study of the blood vessels of dog pulp using corrosion resin casts. Three-dimensional reconstruction of the pulpal vessels.J Endod, 1982; 8:131-135.
  7. Lyroudia K, Nikolaidis N, Pitas I, Zervas P, Palakidis K. Computerized three-dimensional reconstruction: A method to study pulpal vessels and nerves.J Endod, 1993; 19:604-608. [PubMed]
  8. Lyroudia K, Nikolaidis N, Pitas I, Palakidis K. Three computer methods to reconstruct pulpal blood vessels and nerves.J Endod, 1995; 21:501-504.[PubMed]
  9. Karen P, Jirkovská M, Tomori Z, Demjénová E, Janácek J, Kubínová L. Three-dimensional computer reconstruction of large tissue volumes based on composing series of high-resolution confocal images by GlueMRC and LinkMRC software.Microsc Res Tech, 2003; 62(5):415-422. [PubMed] [Crossref]
  10. Chang YC, Lee DJ, Wang YG. Color-texture segmentation of medical images based on local contrast information. Proc. IEEE Symposium Series on Computational Intelligence, Honolulu, Hawaii, USA, 2007.
  11. Krinidis M, Pitas I. Color texture segmentation based on the modal energy of deformable surfaces.IEEE Transactions on Image Processing, 2009; 18(7):1613-1622. [Web of Science] [Crossref]
  12. Jiang Y, Meng J, Babyn P. X-ray image segmentation using active contour model with global constraints. Proc. IEEE Symposium Series on Computational Intelligence, Honolulu, Hawaii, USA, 2007.
  13. KrinidisS, Nikou C, Pitas I. Reconstruction of serially acquired slices using physics-based modelling.IEEE Transactions on Information Technology in Biomedicine, 2003; 7(4):394-403. [Crossref]
  14. Bors A, Kechagias L, Pitas I. Binary morphological shape-based interpolation applied to 3-D tooth reconstruction.IEEE Transactions on Medical Imaging, 2002; 21(2):100-108. [Crossref] [PubMed]
  15. Nikolaidis N, Pitas I. 3D image processing algorithms. Wiley, 2000.
  16. Zitova B, Flusser J. Image registration methods: A survey.Image and Vision Computing, 2003; 21:977-1000. [Web of Science] [Crossref]
  17. Tait R, Schaefer G, Hopgood AA, Nakashima T. High performance image registration using a distributed blackboard architecture. Proc. IEEE Symposium Series on Computational Intelligence, Honolulu, Hawaii, USA, 2007.
  18. Krinidis S, Nikou C, Pitas I. Alignment of serially acquired slices using a global energy function. In Proceedings of the IEEE Engineering in Medicine and Biology Conference (EMBS ‘01), Istanbul, Turkey, 2001.
  19. Ben-Jemaa R, Schmitt F. A solution for the registration of multiple 3D points sets using quaternions. Lecture Notes in Computer Science. In: Proceedings of the 5th European Conference on Computer Vision (ECCV ’98), Freiburg, Germany. Vol. 2, pp 34-50, 1998.
  20. Sramek M. 20 years of volume rendering. Spring Conference on Computer Graphics, Slovakia, 2006.
  21. Plath N, Knorr S, Goldmann L, Sikora T. Adaptive image warping for hole prevention in 3D view synthesis.IEEE Trans Image Process, 2013; 22(9):3420-3432. [Crossref] [Web of Science] [PubMed]
  22. Steiniger BS, Bubel S, Boeckler W, Lampp K, Seiler A, Jablonski B, Guthe M, Stachniss V. Immunostaining of pulpal nerve fibre bundle/arteriole associations in ground serial sections of whole human teeth embedded in technovit 9100.Cells Tissues Organs, 2013; 198(1):57-65. [Crossref] [Web of Science] [PubMed]
  23. Radlanski FR. Morphogenesis of human tooth primordial: the importance of 3D computer-assisted reconstruction.Int J Dev Biol, 1995; 39:249-256.
  24. Peterková R, Peterka M, Vonesch JL, Ruch JV. Contribution of 3-D computer-assisted reconstructions to the study of the initial steps of mouse odontogenesis.Int J Dev Biol, 1995; 39:239-247.
  25. Witter K, Lesot H, Peterka M, Vonesch JL, Misek I, Peterkova R. Origin and developmental fate of vestigial tooth primordia in the upper diastema of the field vole (Microtus agrestis, Rodentia).Arch Oral Biol, 2011; 50(4):401-409. [Crossref]
  26. Arnold WH, Gaengler P, Kalkutschke L. Three-dimensional reconstruction of approximal subsurface caries lesions in deciduous molars.Clin Oral Invest, 1998; 2(4):174-179. [Crossref]
  27. Arnold WH, Gaengler P, Saeuberlich E. Distribution and volumetric assessment of initial approximal caries lesions in human premolars and permanent molars using computer-aided three-dimensional reconstruction.Arch Oral Biol, 2000; 45(12):1065-1071. [Crossref] [PubMed]
  28. Arnold WH, Meiselbach V. 3-D reconstruction of a human fetus with combined holoprosencephaly and cyclopia.Head Face Med, 2009; 29:5-14.
  29. Sohn WJ, Gwon GJ, An CH, Moon C, Bae YC, Yamamoto H, Lee S, Kim JY. Morphological evidences in circumvallate papilla and von Ebners’ gland development in mice.Anat Cell Biol, 2011; 44(4):274-283. [Crossref] [PubMed]
  30. Smith LC. Basic Confocal Microscopy. In: In situ Hybrization and Immunohistochemistry. Current Protocols in Molecular Biology. Published on line: January 2008 in Wiley Interscience.
Citation Information: Balkan Journal of Dental Medicine. Volume 19, Issue 1, Pages 43–49, ISSN (Online) 2335-0245, DOI: https://doi.org/10.1515/bjdm-2015-0033, July 2015