Effect of cannabinoids on rat tenocytes in culture

Nanette Scutt1, Elizabeth Williamson2, Andy Scutt1. 1Sheffield University, Sheffield, United Kingdom, 2Reading University, Reading, United Kingdom.

At least 20% of the population will suffer some form of tendinitis or tendinosis, and surgical reconstructions of tendons number in the hundreds of thousands per year. Despite this, very little is known of the cell biology underlying their physiology and pathology and the range of therapies available to treat tendinopathies is limited to anti-inflammatory drugs, physiotherapy and surgery. It has recently been shown that cannabinoids stimulate fibroblastic-colony (CFU-f) formation by bone marrow cells indirectly via CB2 receptors (Scutt and Williamson 2007), that the CB2 receptor is associated with human osteoporosis (Karsak et al 2005) and that bone resorption can be increased by tetrahydrocannabinol (THC) via CB1 receptors (Idris et al 2005). As tenocytes and osteoblasts are developmentally related we have also investigated the effects of cannabinoids on rat tenocytes in vitro.

Tenocytes were obtained from the tail, Achilles or patella tendons of male 200 g Wistar rats by overnight collagenase digestion. Cells were either plated out in 24 well plates (10,000 cells/cm2) for the determination of NO synthesis, proliferation or collagen accumulation, or in Petri dishes (1000 cells per dish) for the determination of CFU-f.

Neither THC nor 2-AG had any effect on tenocyte proliferation, collagen accumulation or colony formation except at 10−5 M where a small but significant inhibitory effect (12%, p < 0.05) was observed. In contrast, THC and 2-AG dose-dependently inhibited the induction of NO synthesis by IL-1 beta by up to 80% (p < 0.01) at 10-5 M. Attempts to characterise the mechanism of action of THC and 2-AG proved inconclusive. The CB2 agonist JWH 015 had no effect on NO synthesis by IL-1 beta at a dose of 10 μM and in this system, the CB2 selective antagonist AM630 behaved as an agonist, producing a supplementary additive effect to that of THC and 2-AG. The vanilloid agonist resiniferatoxin had no effect in this system and the vanilloid antagonist 5’-iodoresiniferatoxin was ineffective in blocking the effects of THC or 2-AG. Preliminary attempts to identify the cannabinoids receptors by flow cytometry also proved inconclusive. These data show that the cannabinoids THC and 2-AG have potential anti-inflammatory effects in rat tenocytes, but it appears that these activities are not mediated via CB1, CB2 or vanilloid receptors, and the mechanism of action remains to be elucidated. Despite this, due to their comparative lack of side effects, cannabinoids may prove to be an interesting potential alternative anti-inflammatory therapy when compared with existing drugs.

Idris AI, van ‘t Hof RJ, Greig IR, et al. Regulation of bone mass, bone loss and osteoclast activity by cannabinoid receptors. Nat Med. 2005;11:774-779

Karsak M, Cohen-Solal M, Freudenberg J, et al. Cannabinoid receptor type 2 gene is associated with human osteoporosis. Hum Mol Genet. 2005;14:3389-3396

Scutt AM, Williamson EW. Cannabinoids stimulate fibroblastic-colony formation by bone marrow cells indirectly via CB-2 receptors. Calc. Tiss Int. 2007; 80(1):50-9.