A Comparative Analysis of the Osteogenic Potential of Dental Mesenchymal Stem Cells

Abstract

The aim of this study was to compare the in vitro osteogenic differentiation potential of within-subject mesenchymal stem cells (MSCs) derived from the dental pulp of permanent teeth (dental pulp stem cells-DPSCs), the dental pulp of deciduous teeth (stem cells from human exfoliated deciduous teeth-SHEDs), and the periodontal ligament of permanent teeth (periodontal ligament stem cells-PDLSCs). A single subject was identified that required concurrent removal of both deciduous and permanent teeth for orthodontic purposes. Primary, mixed population cells from dental pulp, deciduous dental pulp, and periodontal ligament were obtained by the tissue outgrowth method. Subsequently, isolation of STRO-1 +ve cells from their respective primary cell cultures was achieved by immunomagnetic separation. Cells were induced with an osteogenic cocktail of 5 mM β-glycerophosphate, 100 nM dexamethasone, and 50 mg/mL ascorbic acid for up to 21 days. Osteogenic responses were assessed functionally by an alkaline phosphatase (ALP) activity assay and an alizarin red staining assay. Expression of the early osteogenic associated genes, alkaline phosphatase gene (ALPL), runt-related transcription factor 2 (RUNX2), collagen type I alpha 1 (COL1A1), and secreted phosphoprotein 1 (SPP1), was compared by qPCR at days 1, 4, and 7 of differentiation. Functional analysis revealed that there were significant differences in intracellular ALP activity on days 4, 7, 10, and 14 with PDLSCs > SHEDs > DPSCs. Quantification of alizarin red staining showed significantly more mineralization for PDLSCs by day 21. Gene expression analysis showed significant early upregulations of the osteogenic markers ALPL and COL1A1 for PDLSCs over DPSCs and SHEDs. SHEDs showed significantly higher upregulation of ALPL over DPSCs. In conclusion, PDLSCs showed a significantly higher osteogenic differentiation potential than both DPSCs and SHEDs evidenced by functional studies and gene expression. This may be of significance for the use of dentally derived MSCs in bone tissue engineering applications.

Keywords: alizarin red staining; alkaline phosphatase; osteoblastic differentiation; periodontal ligament stem cells; qPCR.