Objective: The study aimed to investigate acetylsalicylic acid (ASA) effects on osteo/odontogenic differentiation and proliferation of dental pulp stem cells (DPSCs) in vitro and the potential involvement of adenosine monophosphate-activated protein kinase (AMPK) pathway in these processes.
Design: DPSCs were isolated from third molars pulp tissues of five patients and grown in osteogenic medium alone or supplemented with ASA. Expression of DPSCs markers was tested by flow-cytometry. Cytotoxicity of ASA at concentrations of 10, 50 and 100 µg/ml was tested by MTT and NR assays. Osteo/odontogenic differentiation was analyzed via alizarin red staining and ALP activity. Quantitative PCR (qPCR) was used for osteo/odontogenic markers' (DSPP, BMP2, BMP4, BSP, OCN and RUNX2) and c-Myc expression analysis. AMPK inhibition of ASA-induced osteo/odontogenesis was tested by qPCR of selected markers (DSPP, OCN and RUNX2).
Results: Cytotoxicity assays showed that only the highest ASA dose decreased cell viability (89.1 %). The smallest concentration of ASA applied on DPSCs resulted in a remarkable enhancement of osteo/odontogenic differentiation, as judged by increased mineralized nodules' formation, ALP activity and gene expression of analyzed markers (increase between 2 and 30 folds), compared to untreated cells. ASA also increased DPSCs proliferation. Interestingly, AMPK inhibition per se upregulated DSPP, OCN and RUNX2; the gene upregulation was higher when ASA treatment was also included. c-Myc expression level decreased in cultures treated with ASA, indicating undergoing differentiation processes.
Conclusions: Low concentrations of ASA (corresponding to the standard use in cardiovascular patients), were shown to stimulate osteo/odontogenic differentiation of dental pulp stem cells.
Keywords: Acetylsalicylic acid; Adenosine monophosphate-activated protein kinase (AMPK); Dental pulp stem cells; Osteogenic/odontogenic differentiation; Proliferation.
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