Abstract
Primary tooth pulp cells have a unique character that can be attributed to their neural crest origin. The long-term aim of this research is to utilise these cells to regenerate dentine–pulp complexes in damaged immature permanent teeth. In the first instance, it is necessary to successfully culture and characterise progenitor cells isolated from primary dental pulp and assess their ability to differentiate into cells of different origins. Future research can proceed to treatment-based clinical trials using ‘progenitor cells’ from primary teeth to treat future problems or diseases that may arise in the same patient by regenerating the pulp–dentine complex, leading to root maturation; and potentially regenerating damaged heart muscle or nerves.
Objectives
To characterise primary pulp cells from three defined stages of root resorption by in vitro immunocytochemistry and immunohistochemistry experiments. The expression of markers for embryonic, neural and mesenchymal stem cells were studied. The differentiation potential of primary pulp cells into the three germ layer lineages, specialised mesoderm (cardiomyocyte progenitor cells) and specialised ectoderm (neural progenitor cells) was assessed.
Methods
Caries-free primary canines at three defined stages of physiological root resorption were included in this study (n = 9). In vitro cultures were established in xeno-free, serum-free Essential 8 (E8) medium on a human truncated vitronectin matrix (VTN-N). An embryonic stem cell line (GENEA002) grown in stem cell media (M2) was used as a positive control in immunocytochemistry and multilineage differentiation experiments. The expression of embryonic stem cell proteins (Oct4A, Nanog and Sox2), neural stem cell proteins (nestin and distal-less homeobox 2 [Dlx2]) and mesenchymal stem cell surface markers (cluster of differentiation markers CD90, CD73 & CD105) was investigated. Flow cytometry analyses were also performed based on criteria proposed by the International Society for Cellular Therapy.
The differentiation potential of primary pulp cells into ectoderm, mesoderm and endoderm, as well as cardiomyocytes and neural progenitors, was determined.
Results
Most primary tooth pulp cells in culture and in tissue sections expressed the early neural progenitor markers nestin and Dlx2; a few isolated cells expressed the embryonic stem cell transcription factors Oct4A, Nanog and Sox2; and the majority expressed the mesenchymal stem cell markers CD90, CD105 and CD73. Flow cytometry for mesenchymal stem cell markers did not detect differences according to resorption stage.
Differentiated cells showed positive immunofluorescence for orthodenticle homeobox-2 (Otx2) (ectoderm), Brachyury (mesoderm) and Sox17 (endoderm) proteins of the three germ layers as well as expression of cardiomyocyte progenitor markers TNNT2 and Nkx2.5 and neural progenitor markers nestin, Sox1, Sox2 and Pax6.
Conclusions
The present study identified cells that variably express marker proteins belonging to embryonic stem cells, neural stem cells and mesenchymal stem cells. The in vitro ability of the cells to differentiate into the three germ layers cells, as well as cardiomyocyte and neural progenitor cells, was detected. The root resorption stage was not a significant factor in relation to mesenchymal stem cell marker expression.
This research provides an insight into the unique type of progenitor cells present in the dental pulp of primary teeth and their differentiation capabilities that can be utilised to regenerate the pulp–dentine complex in damaged permanent teeth following injury.