Isolation, Identification And Biological Characterization Of Rat Dental Pulp Stem Cells

In recent years, scientists have discovered a wide array of stem cells that have unique capabilities to self-renew, grow indefinitely, and differentiate or develop into multiple types of cells and tissues. These cells can be divided into two groups according to their origin, namely embryonic stem cells originated from the inner cell mass of blastocyst and adult stem cells that exist in various adult tissues. Many adult stem cells have been identified and successfully isolated from various tissues including bone marrow, muscle, neural tissue and skin. Dental pulp stem cell was first isolated by Dr. Gronthos and his colleagues in year 2000 from human dental pulp. Dental pulp stem cells are similar to other adult stem cells so far have been identified. They have great potential of self-replication and can differentiate into functional mature cell types or trans-differentiate into other cell types. Though successful isolation of human dental pulp stem cells has been reported, investigations on rat dental pulp cells have not yet been documented. As one of the most frequently used animal models in scientific research, rat also plays an important role in dental research because it's less costly and of high homogeneity in comparison with human beings. In this study, rat dental pulp stem cell clones were isolated from cultured pulp cells and the isolated clones were characterized through a series of biological examinations. Part I Isolation and identification of rat dental pulp stem cellsThough human dental pulp stem cells have been successfully isolated, references relating to the cell isolation methods are rare. In this study, single cell clones generated from dental pulp tissue were culture-expanded and induced in vitro or in vivo to differentiate into various cell types. Cells originated from 2B2 clone were found to have a long protrude just like an odontoblast after induction by mineralization solution. At the same time, odontoblast-specific DSPP expression was also detected. After 8 weeks of subcutaneous implantation, the2B2 cells was found to form dentin-pulp complex structure with layers of mineralized dentin, predentin, odontoblast and pulp cells, which is similar to normal dentin-pulp complex structure.Mature method for identification of dental pulp stem cells has not been well-developed yet. In many tissues, adult stem cells and progenitor cells are difficult to be distinguished from each other and thus were named ambiguously. Present opinions tend to regard purified candidate stem cells as cells that can re-establish tissue, while progenitors as cells that can only differentiate along a committed lineage. The formation of dentin-pulp complex is a potent evidence of rat dental pulp stem cell. The dentin-pulp complex, which originates from ectoderm, is unique in tooth development. The intact dentin-pulp complex structure developed from in vivo implanted 2B2 cells contains layers of mineralized dentin, pre-dentin, odontoblast and pulp cells. The potential of in vitro differentiation and the formation of dentin-pulp complex structure provide sufficient evidence for the identification of rat dental pulp cell. Part II Biological analysis of rat dental pulp stem cellThe biological characteristics of the first-time isolated rat dental pulp stem cell was conducted, including cell morphology, growth and proliferation, mineralization and clone formation capacity. Cell cycle and karyotype analysis were also investigated to make a comprehensive understanding of the 2B2 cells. Morphology study provides basic but important information for us to know a new cell. The 2B2 rat dental stem cells we obtained grow and proliferate like fibroblasts. Polygonal or ellipse cells can be seen in high-density areas. As indicated by the growth curve, 2B2 cells are a bit less vigorous than rat bone marrow-derived mesenchymal stem cells, with a mean doubling time of 58.3 hours and reaching a maximum of OD at day 8-10. The CFU-F of 2B2 cells is 2.55%, higher than the primary rat dental pulp cells. The 2B2 cells also demonstrated norm...