Cell and Tissue Research

, Volume 353, Issue 1, pp 65–78 | Cite as

Effect of isolation methodology on stem cell properties and multilineage differentiation potential of human dental pulp stem cells

  • P. Hilkens
  • P. Gervois
  • Y. Fanton
  • J. Vanormelingen
  • W. Martens
  • T. Struys
  • C. Politis
  • I. Lambrichts
  • A. BronckaersEmail author
Regular Article


Dental pulp stem cells (DPSCs) are an attractive alternative mesenchymal stem cell (MSC) source because of their isolation simplicity compared with the more invasive methods associated with harvesting other MSC sources. However, the isolation method to be favored for obtaining DPSC cultures remains under discussion. This study compares the stem cell properties and multilineage differentiation potential of DPSCs obtained by the two most widely adapted isolation procedures. DPSCs were isolated either by enzymatic digestion of the pulp tissue (DPSC-EZ) or by the explant method (DPSC-OG), while keeping the culture media constant throughout all experiments and in both isolation methods. Assessment of the stem cell properties of DPSC-EZ and DPSC-OG showed no significant differences between the two groups with regard to proliferation rate and colony formation. Phenotype analysis indicated that DPSC-EZ and DPSC-OG were positive for CD29, CD44, CD90, CD105, CD117 and CD146 expression without any significant differences. The multilineage differentiation potential of both stem cell types was confirmed by using standard immuno(histo/cyto)chemical staining together with an in-depth ultrastructural analysis by means of transmission electron microscopy. Our results indicate that both DPSC-EZ and DPSC-OG could be successfully differentiated into adipogenic, chrondrogenic and osteogenic cell types, although the adipogenic differentiation of both stem cell populations was incomplete. The data suggest that both the enzymatic digestion and outgrowth method can be applied to obtain a suitable autologous DPSC resource for tissue replacement therapies of both bone and cartilage.


Dental pulp stem cells Isolation method Mesenchymal trilineage differentiation Ultrastructural analysis 

Supplementary material

441_2013_1630_Fig8_ESM.jpg (61 kb)
Supplementary Fig. 1

Adipogenic differentiation of DPSC-EZ and DPSC-OG. Ultrastructurally, adipogenic differentiated cells are globularly shaped (f) with a cytoplasm filled with electron-dense vesicles (v in figure g and h), branched mitochondria (h, bMit) and dilated rough endoplasmatic reticulum (i, dER). Scale bars: a = 10 μm; b, c, d = 2 μm (JPEG 61 kb)

441_2013_1630_MOESM1_ESM.tif (8.8 mb)
High resolution image (TIFF 9009 kb)
441_2013_1630_Fig9_ESM.jpg (34 kb)
Supplementary Fig. 2

TEM analysis of 3D-chondrogenic differentiated DPSCs. Chondrogenic differentiated DPSC-EZ (a) and DPSC-OG (d) both display an elongated cell phenotype with a cytoplasm containing numerous ribosomes and electron-dense intracellular matrix vesicles (mv, figure e). Striated collagen fibers (cf) are abundant in the ECM (b). Cartilage fragments (ca) are present in the ECM of both cell types and are closely interacting with the surrounding DPSCs and ECM (c, f). Scale bars: a, d, e = 5 μm; b = 1 μm; c = 10 μm; f = 2 μm (JPEG 33 kb)

441_2013_1630_MOESM2_ESM.tif (4.1 mb)
High resolution image (TIFF 4213 kb)


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Copyright information

© Springer-Verlag Berlin Heidelberg 2013

Authors and Affiliations

  • P. Hilkens
    • 1
  • P. Gervois
    • 1
  • Y. Fanton
    • 1
  • J. Vanormelingen
    • 1
  • W. Martens
    • 1
  • T. Struys
    • 1
  • C. Politis
    • 1
    • 2
  • I. Lambrichts
    • 1
  • A. Bronckaers
    • 1
    Email author
  1. 1.Department of Functional Morphology, Laboratory of Histology, Biomedical Research Institute (BIOMED)Hasselt UniversityDiepenbeekBelgium
  2. 2.Department of Oral and Maxillofacial Surgery, KU LeuvenUniversity Hospitals LeuvenLeuvenBelgium

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