Adenosine A1 receptor stimulation enhances osteogenic differentiation of human dental pulp-derived mesenchymal stem cells via WNT signaling

In this study, mesenchymal stem cells deriving from dental pulp (DPSCs) of normal human impacted third molars, previously characterized for their ability to differentiate into osteoblasts, were used. We observed that: i) DPSCs, undifferentiated or submitted to osteogenic differentiation, express all four subtypes of adenosine receptors (AR) and CD73, corresponding to 5'-ecto-nucleotidase; and ii) AR stimulation with selective agonists elicited a greater osteogenic cell differentiation consequent to A(1) receptor (A(1)R) activation. Therefore, we focused on the activity of this AR. The addition of 15-60 nM 2-chloro-N-6-cyclopentyl-adenosine (CCPA), A(1)R agonist, to DPSCs at each change of the culture medium significantly increased the proliferation of cells grown in osteogenic medium after 8 days in vitro (DIV) without modifying that of undifferentiated DPSCs. Better characterizing the effect of A(1)R stimulation on the osteogenic differentiation capability of these cells, we found that CCPA increased the: i) expression of two well known and early osteogenic markers, RUNX-2 and alkaline phosphatase (ALP), after 3 and 7 DIV; ii) ALP enzyme activity at 7 DIV and iii) mineralization of extracellular matrix after 21 DIV. These effects, abolished by cell pre-treatment with the A(1)R antagonist 8-cyclopentyl-1,3-dipropyl-xanthine (DPCPX), involved the activation of the canonical Wnt signaling as, in differentiating DPSCs, CCPA significantly increased dishevelled protein and inhibited glycogen synthase kinase-3 beta, both molecules being downstream of Wnt receptor signal pathway. Either siRNA of dishevelled or cell pre-treatment with Dickkopf-1, known inhibitor of Wnt signaling substantially reduced either DPSC osteogenic differentiation or its enhancement promoted by CCPA. Summarizing, our findings indicate that the stimulation of A(1)R may stimulate DPSC duplication enhancing their osteogenic differentiation efficiency. These effects may have clinical implications possibly facilitating bone tissue repair and remodeling. (C) 2013 Elsevier B.V. All rights reserved.