Abstract
Purpose: To isolate and characterize mesenchymal stem cells of dental follicle from fresh and cryopreserved samples and to test any significant difference in their osteogenic differentiation potential by using digital imaging software. We also investigated whether the cryoprotectant used and its concentration is able to maintain cell count and viability. Methods: Mesenchymal stem cells (MSCs) were isolated from dental follicle of impacted third molars. The osteogenic differentiation potential of dental follicle stem cells was assessed using alizarin red and alkaline phosphatase staining followed by digital imaging quantification of the stains. Results: Dental follicle cells have shown typical characterisation by exhibiting the stem cell stromal markers and hematopoietic markers, but there was variance in the percentage of expression in fresh and cryopreserved samples. There was considerable osteogenic differentiation potential in the fresh sample compared to cryopreserved sample. The cell count and viability were preserved in both samples. Conclusions: The results in the study have shown wide variation of osteogenic differentiation potential in fresh and cryopreserved samples. Also, the cryoprotectant was found to be effective in its purpose at the specified concentration.
Highlights
Mesenchymal stem cells (MSCs) have proved to be invaluable in regenerative medicine because of their two remarkable characteristics of self-renewal and multi-lineage differentiation
The Dental follicle stem cells (DFSCs) manifest characteristics of both mesoderm and ectoderm, which contributes to the union of mesenchymal stem cells and epithelial stem cells, which is imperative for regenerating a new tooth[2,12]
Cells were positive and viable in cryopreserved samples, cell count had decreased to an average of 0.58 X 106 cells/ml, as they were stored in 2 separate vials while being frozen, so they were subjected to subculture before undertaking characterisation and differentiation
Summary
Mesenchymal stem cells (MSCs) have proved to be invaluable in regenerative medicine because of their two remarkable characteristics of self-renewal and multi-lineage differentiation. The ASCs isolated from various dental tissues are commonly referred to as Dental Stem Cells (DSCs). The dental follicle is a loose connective tissue that surrounds the developing tooth and is separated from dentin by an epithelial layer (Hertwig’s sheath) with three distinct stem cell populations (hDF1, hDF2, hDF3), distinct morphologies, gene expression and differentiation potential[6,7]. DFSCs can be isolated from developing tissues, compared to other stem cells of dental tissue origin[9]. DFSCs exhibit fibroblastic morphology and excellent proliferative capacity, with selective adherence to solid surfaces. They can be cryopreserved for either research or future medical therapy. There is controversy regarding the optimal cryoprotective agent and the concentration required to minimise cytotoxicity of the banked cells, the minimum number of cells required for cryopreservation to obtain viable cell lines after thawing, the storage time and the storage temperature
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