Abstract
Category:
Basic Sciences/Biologics; Basic Sciences/Biologics; Sports; Other
Introduction/Purpose:
Recently, purposes of medicinal marijuana including analgesia and reduction in inflammation. The active component activates the CB1 and CB2 receptors, thus mimicking the action of endogenous cannabinoids. CB1 and CB2 are important regulators of bone metabollism. In particular, endocannabinoid signaling has shown to regulate proliferation and differentiation of MSCs and the activities of osteoblasts and osteoclasts. Cannabinoids may be prescribed to patients suffering bone fractures involving prolonged immobilization and discomfort. Thus, there is an important need to understand more completely the role of cannanbinoid signaling in fracture healing. In this study, we examine the influence of a synthetic cannabinoid agonist, Win-55,212-2 (Win) on adult, human MSC-derived osteoblast activity. Cell viability and osteogenic phenotype were assessed by MTS assay, RT-PCR, and alizarin red staining.
Methods:
With an IRB approved protocol, MSCs were isolated from healthy human bone marrow and expanded to the passage five for experimentation. Cells were then plated at 5,200 cells/cm2 (6-well plate) and osteogenically stimulated for 21 days before treatment with increasing concentrations of Win (0.01, 0.1, or 1 µM) for an additional 48 hours. The MTS assay was employed to determine the half-maximal (50%) inhibitory concentration (IC50) at different time points. The cell phenotype was assessed by real-time PCR and alizarin red staining.
Results:
We first used the MTS assay (for metabolism and proliferation) to test 7 doses of Win-55, from 0.001µM up to 5 µM and determined that the human osteoblast IC50 of Win-55 over 1-7 days of exposure was ~ 1.5 µM (Figure 1), a dose slightly lower than concentrations found to affect chondrocytes in a previous study. At 1µM Win-55, RT-PCR revealed that the expression of inflammatory cytokines IL-1beta and TNF-alpha was significantly reduced, while that of IL-6 and COX2 was unchanged. The expression of most osteogenic markers was unchanged by Win, but we did observe significant increases in BMP2, RUNX2 and OPN (Figure 2). Alizarin Red staining revealed increased calcium deposition and mineralization in the presence of 1µM Win, corroborating the RT-PCR results (Figure 3).
Conclusion:
Win-55,212-2 may be beneficial to osteogenesis. 1 µM Win enhanced the osteoblastic phenotype over control, untreated MSC-derived osteoblasts, reduced inflammatory mediators and increased calcium deposition and mineralization. This suggests that cannabidiol treatment might lead to improvements in fracture healing and provide a novel therapeutic option for the bone regeneration. Future studies are aimed at characterizing the CB-1 and CB-2 signaling associated with the changes observed here and the effects of Win on MSC-derived osteoblasts in the context of inflammation.
