| Summary: | Introduction: The endocannabinoid signalling system has been implicated in a broad spectrum of pathological and physiological processes including the control of inflammation and pain. Recent pre-clinical studies have demonstrated the therapeutic potential of cannabis-based drugs in the treatment of inflammatory musculoskeletal conditions, including rheumatoid arthritis. Studies in rodent models suggest that the endocannabinoid system modulates bone remodelling, via actions at the cannabinoid CB1, CB2 receptors and the TRPVl ion channel. The effects of the endocannabinoid system in inflammatory processes and bone homeostasis suggest that they may impact on the pathogenesis of Charcot osteoarthropathy, a non-infective, chronic destructive condition of the bone initiated by neuro-traumatic stimuli, most prevalent in patients with diabetic peripheral neuropathy. Neuropathy-induced changes in the endocannabinoid system may contribute to bone breakdown in Charcot, dependent or independent of the RANKL/RANK/OPG signalling pathway which is known to regulate bone remodelling. The aims of this thesis were to quantify and compare the levels of endocannabinoids and components of the endocannabinoid receptor system in human trabecular bone from Charcot and non-Charcot subjects, to identify a functional endocannabinoid system in human osteoclasts in culture, and to determine the effect of TRPVl modulation on osteoclastogenesis. Methods: Bone biopsies and blood were taken from patients with Charcot osteoarthropathy or those undergoing elective foot surgery. Histological features of the Charcot and non-Charcot bone were compared following staining with haematoxylin. Plasma levels of the inflammatory marker, CRP, and bone turnover markers (ALP, CTx) were measured with routine biochemical analysis. Plasma levels of RANKL, OPGJ and inflammatory cytokines (TNFa, IL-l, IL-6, IL-7) were measured using enzyme-linked immunosorbent assay. Levels of endocannabinoids in plasma and bone were analysed by liquid chromatography-tandem mass spectrometry (LC- MS/MS). Osteoclasts were differentiated from U-937 cells (human leukaemic monocyte lymphoma cells) using TPA (O.Lpq/rr»). followed by calcitriol (10- BM) or TNFa (3ng/ml). Tartrate-resistant acid phosphatase (TRAP) staining and resorption of calcium phosphatase were used to identify mature osteoclasts. Components of the endocannabinoid system, together with osteoclast markers (TRAP, cathepsin K) and RANKL/OPG were detected at the mRNA level in human trabecular bone and osteoclasts with quantitative PCR. Modulation of ATP(lOOIJM)-induced calcium responses by CB1 and CB2 receptor agonists (ACEA (lIJM), JWH133 (3IJM), CP55940 (3IJM» were used to demonstrate the presence of functional cannabinoid receptors. Exposure of osteoclasts to the TRPVl receptor antagonist capsazepine (3IJM) and agonist AEA (lIJM) was used to study the role of TRPVl in osteoclastogenesis. Results: Histology of the Charcot bone showed a loss of bone microarchitecture, loss of marrow space and features of increased bone turnover. Higher levels of ALP, CTx and IL-6 were detected in Charcot plasma, compared to controls, indicating an inflammatory response and an increase in bone turnover. An increase in OPG level, but not RANKL was found in Charcot plasma. The endocannabinoids 2-AG, AEA and related compounds PEA and OEA were detected in human trabecular bone (not AEA) and plasma although no significant differences were evident between Charcot and control samples. Components of the endocannabinoid system, with the exception of CB2 receptors, were detected at the mRNA level in human trabecular bone. Lower levels of the endocannabinoid-related nuclear receptor PPARy (known to be anti-inflammatory) were found in Charcot bone compared to that of controls. Osteoclasts differentiated from U-937 cells showed positive TRAP staining, were multinucleated and calcium phosphate-resorbing from day 5 of culture. mRNA expression of components of the endocannabinoid system were significantly up-regulated with osteoclast maturation. The application of CB1/2 receptor agonists modified ATP-induced calcium responses in osteoclasts. Exposure of osteoclasts to capsazepine, but not AEA, Significantly attenuated the number of TRAP-positive osteoclasts. Effects of capsazepine were attenuated by AEA. Conclusion: Work in this thesis has confirmed the link between Charcot and an increase in bone turnover (increased ALP and CTx levels), and the presence of an inflammatory response (increased IL-6 levels). It also demonstrated the presence of active endocannabinoid signalling, in Charcot and non-Charcot trabecular bone, and osteoclasts. Marked histological changes were observed in Charcot bone. The lack of genetic data and the sporadic and rare nature of this condition renders it difficult to establish the mechanisms of the disease. Marked differences were seen in the effect of osteoclast maturation on the endocannabinoid system. Importantly, the process of osteoclastogenesis was seen to be halted by TRPVl channel inhibition suggesting that pharmacological interventions targeting TRPVl could be used in the control of osteoclastogenesis. These data add to the growing evidence that the endocannabinoid signalling system plays an important role in bone homeostasis, and that alterations in the components of this system may contribute to pathological conditions of the bone, such as osteoporosis, osteoarthritis, and Paget's disease.
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