Cannabinoids Stimulate Fibroblastic Colony Formation by Bone Marrow Cells Indirectly via CB₂ Receptors

Recently, the cannabinoid receptors CB₁ and CB₂ were shown to modulate bone formation and resorption in vivo, although little is known of the mechanisms underlying this. The effects of cannabinoids on mesenchymal stem cell (MSC) recruitment in whole bone marrow were investigated using either the fibroblastic colony-forming unit (CFU-f) assay or high-density cultures of whole bone marrow. Levels of the CB₁ and CB₂ receptors were assessed by flow cytometry. Treatment of CFU-f cultures with the endocannabinoid 2-arachidonylglycerol (2-AG) dose-dependently increased fibroblastic and differentiated colony formation along with colony size. The nonspecific agonists CP 55,940 and WIN 55,212 both increased colony numbers, as did the CB₂ agonists BML190 and JWH015. The CB₁-specific agonist ACEA had no effect, whereas the CB₂ antagonist AM630 blocked the effect of the natural cannabinoid tetrahydrocannabivarin, confirming mediation via the CB₂ receptor. Treatment of primary bone marrow cultures with 2-AG stimulated proliferation and collagen accumulation, whereas treatment of subcultures of MSC had no effect, suggesting that the target cell is not the MSC but an accessory cell present in bone marrow. Subcultures of MSCs were negative for CB₁ and CB₂ receptors as shown by flow cytometry, whereas whole bone marrow contained a small population of cells positive for both receptors. These data suggest that cannabinoids may stimulate the recruitment of MSCs from the bone marrow indirectly via an accessory cell and mediated via the CB₂ receptor. This recruitment may be one mechanism responsible for the increased bone formation seen after cannabinoid treatment in vivo.