Background: Endocannabinoids, lipid mediators derived from arachidonic acids, are found to be associated with multiple regulatory functions in several tissues. The endocannabinoids anandamide (AEA) and 2-arachidonoylglycerol (2-AG) are produced within bone microenvironment, and EC system has recently been implicated in the regulation of bone metabolism. Human periodontal ligament stem cells (hPdLSCs) are characterised as having multipotent differentiation properties and represents postnatal stem cell sources for regenerative and immunomodulatory therapies. In the present study, the effects of AEA and 2-AG were evaluated on multi-lineage differentiation potentials and related gene expression of hPdLSCs in different designed culture conditions.
Methods: Surface expression of characteristic markers in hPdLSCs was analysed by flow cytometry. Osteogenic and adipogenic differentiation potential of hPdLSCs was evaluated by Alizarin Red and Oil Red O staining, respectively. The proliferation/viability of hPdLCs was measured by 3,4,5-dimethylthiazol-2-yl-2,5-diphenyl tetrazolium bromide (MTT) assay. Gene expression analyses of runt-related transcription factor 2 (RUNX2), osteocalcin (OCN) and collagen type 1 (COL1) by real-time PCR.
Results: hPdLSCs exhibited positive staining to mesenchymal markers CD29, CD90, CD105, CD146 and negative staining to haematopoietic markers CD14, CD34, and CD45 as well as the ability to differentiate in osteoblasts and adipocytes. AEA and 2-AG did not reveal any significant effects on proliferation/viability of hPdLSCs. AEA (10 μM) significantly increased COL1 gene expression (p<0.05), whereas 2-AG (10 μM) stimulation resulted with significant up-regulation of RUNX2 and COL1 (p<0.05). When both endocannabinoids (10 μM, each) stimulated hPdLSCs, significant up-regulation of RUNX2 was observed (p<0.05).
Conclusion: AEA and 2-AG appears to have an important modulatory effect on osteogenic differentiation of hPdLSCs. Further studies are needed to elucidate the role of cannabinoid system on hPdLSCs differentiation potential and to evaluate the therapeutic potential of pharmacological modulation of cannabinoid system.
Disclosure: The authors declared no competing interests. The study was supported by Authors Institution (Medical University of Vienna).