Abstract
The dental pulp represents an easily accessible source of adult dental pulp stem cells (DPSCs). The purpose of this study was to optimize the isolation protocol for DPSCs, namely to shorten the time of enzymatic digestion of the dental pulp, and to cultivate isolated DPSCs using this new approach, investigate their proliferation, phenotype, cell viability and determine their ability to differentiate into mature cells, chondroblasts, osteoblasts, and adipocytes.
Out of five extracted permanent teeth, we isolated five dental pulp stem cell lineages. They were cultivated in a modified cultivation media (α-MEM) for mesenchymal adult progenitor cells containing 2 % fetal bovine serum (FBS) and supplemented with growth factors, antibiotics, antimycotics and Insulin-Transferrin-SodiumSelenium supplement (ITS).
The cell viability, cell count and other properties were examined using a ViCell analyzer and Z2-Counter. The phenotype analysis was performed using a flow cytometer Cell Lab Quanta.
For differentiation in chondroblasts, osteoblasts and adipocytes, we used commercially available differentiation media. The evidence of differentiation was proved by the immunocytochemistry (osteocalcin and collagen type II) and histological staining (blue Masson's trichrome, von Koss stain and oil red).
We were able to cultivate DPSCs over 47.8 +- 2.0 population doublings (PD). The average population doubling time (DT) was 39.2 +- 6.1 hours.
The average cell viability was 92.3 +- 1.5 % in the second passage and 92.4 +- 1.4 % in the eighth passage. DPSCs showed high positivity for mesenchymal stem cell markers (cluster of differentiation; CD) CD29, CD44, CD90 and for stromal associated markers CD13, CD73, CD166 and negative expression or low positivity for hematopoietic markers CD34, CD45 and for CD31.
DPSCs differentiated into osteoblasts and chondroblasts. Even after the exposition of the strong adipogenic medium they did not show any signs of differentiation into adipocytes.
We have successfully optimized the isolation protocol for DPSCs by shortening the time of enzymatic digestion of the dental pulp. DPSCs isolated using the new method demonstrated the high proliferation and differentiation potential throughout long-term cultivation.
We did not observe any signs of spontaneous differentiation or cell degeneration. DPSCs seems to be the promising future for a regenerative and reparative medicine Thanks to their remarkable high proliferative potential and ability to differentiate into many mature cell populations.