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Semi-quantitative monitoring of confluence of adherent mesenchymal stromal cells on calcium-phosphate granules by using widefield microscopy images

  • Filippo Piccinini
  • Michela Pierini
  • Enrico Lucarelli
  • Alessandro BevilacquaEmail author
Article

Abstract

The analysis of cell confluence and proliferation is essential to design biomaterials and scaffolds to use as bone substitutes in clinical applications. Accordingly, several approaches have been proposed in the literature to estimate the area of the scaffold covered by cells. Nevertheless, most of the approaches rely on sophisticated equipment not employed for routine analyses, while the rest of them usually do not provide significant statistics about the cell distribution. This research aims at studying confluence and proliferation of mesenchymal stromal cells (MSC) adherent on OSPROLIFE®, a commercial biomaterial in the form of granules. In particular, we propose a Computer Vision approach that can routinely be employed to monitor the surface of the single granules covered by cells because only a standard widefield fluorescent microscope is required. In order to acquire significant statistics data, we analyse wide-area images built by using MicroMos v2.0, an updated version of a previously published software specific for stitching brightfield and phase-contrast images manually acquired via a widefield microscope. In particular, MicroMos v2.0 permits to build accurate “mosaics” of fluorescent images, after correcting vignetting and photo-bleaching effects, providing a consistent representation of a sample region containing numerous granules. Then, our method allows to make automatically a statistically significant estimate of the percentage of the area of the single granules covered by cells. Finally, by analysing hundreds of granules at different time intervals we also obtained reliable data regarding cell proliferation, confirming that not only MSC adhere onto the OSPROLIFE® granules, but even proliferate over time.

Keywords

Ground Truth Mesenchymal Stromal Cell Cell Segmentation Cell Confluence High Confluence 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Notes

Acknowledgments

The authors thank Eurocoating S.p.A. for providing OSPROLIFE® HA-TTCP granules; Davide Donati (Osteoarticular Regeneration Laboratory, 3rd Clinic of Orthopaedics and Traumatology, Rizzoli Orthopaedic Institute, Bologna, Italy) for performing the bone marrow harvest; Jennifer Perugini (University of Notre Dame, Notre Dame, Indiana, USA) for English revision of the manuscript; Panagiota Dimopoulou (Osteoarticular Regeneration Laboratory, Rizzoli Orthopaedic Institute) for editorial assistance.

Conflict of interest

The authors declare that they have no conflict of interest and no competing financial interests.

Supplementary material

10856_2014_5242_MOESM1_ESM.zip (1.1 mb)
Supplementary material 1 (ZIP 1142kb)

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Copyright information

© Springer Science+Business Media New York 2014

Authors and Affiliations

  • Filippo Piccinini
    • 1
  • Michela Pierini
    • 2
  • Enrico Lucarelli
    • 2
  • Alessandro Bevilacqua
    • 1
    • 3
    Email author
  1. 1.Advanced Research Center on Electronic Systems for Information and Communication Technologies “E. De Castro” (ARCES)University of BolognaBolognaItaly
  2. 2.Osteoarticular Regeneration LaboratoryRizzoli Orthopaedic InstituteBolognaItaly
  3. 3.Department of Computer Science and EngineeringUniversity of BolognaBolognaItaly

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