Introduction 1 2 3 4 5 6 7 The aims of this study were: (1) to generate functional human osteoclasts in vitro from diabetic patients with acute Charcot’s osteoarthropathy and from healthy and diabetic control participants; (2) to compare the extent of osteoclast formation and resorption; and (3) to determine the role of the RANK/RANKL/OPG pathway in osteoclastic activity in Charcot’s osteoarthropathy. Methods Patients p p p p 8 Ethical permission for this study was obtained from the King’s College Hospital Research Ethics Committee and all participants gave written informed consent. Isolation and culture of monocytes 6 g g 5 2 Osteoclast formation After 14 days, the coverslips were examined histochemically for the expression of tartrate-resistant acid phosphatase (TRAcP), an osteoclast marker. Coverslips with newly formed osteoclasts were collected and rinsed in PBS buffer, fixed with formalin (10% [vol./vol.] in PBS buffer) for 10 min and rinsed in distilled water. TRAcP was histochemically revealed by a simultaneous coupling reaction using Naphtol AS-BI-phosphate as substrate and Fast violet B as the diazonium salt. The coverslips were incubated for 90 min at 37°C in a dark room, rinsed three times in distilled water and the residual activity was inhibited by 4% NaF (wt/wt) for 30 min. Coverslips were then rinsed in distilled water, counterstained with DAPI for 20 min and allowed to dry before mounting, using an aqueous medium. TRAcP-positive cells with more than three nuclei were identified as osteoclasts. The number of newly generated osteoclasts was assessed using a light microscope examination. Osteoclast resorption 4 Statistical analyses U U p Results Osteoclast cultures in the presence of M-CSF Osteoclast formation p p p Osteoclast resorption p p p Osteoclast cultures in the presence of M-CSF and sRANKL Osteoclast formation p 1 p p p p p 2 Fig. 1 a c e b d f a b c d e f  Fig. 2 a n b U a p p p b p p p  Osteoclast resorption p p p 1 p p p 2 Osteoclast cultures in the presence of M-CSF, sRANKL and excess concentrations of OPG Osteoclast formation p p p p p p 3 Fig. 3 a n b U a p p p b p p p  Osteoclast resorption p p p 3 p p p Discussion This study shows that monocytes from patients with acute Charcot’s osteoarthropathy cultured in the presence of M-CSF alone were capable of differentiating into mature osteoclasts that exhibited increased resorption compared with diabetic and healthy control participants. Furthermore, osteoclasts generated after the addition of sRANKL were functionally more aggressive, exhibiting a considerable increase in the extent of resorbing activity in patients with acute Charcot’s osteoarthropathy. This resorption was partially blocked by the addition of excess concentrations of OPG, a soluble receptor decoy for RANKL. This suggests that the increased osteoclastic activity in patients with acute Charcot’s osteoarthropathy is mediated through both a RANKL-dependent and a RANKL-independent pathway. 9 10 11 12 13 14 15 16 15 11 12 13 14 15 17 18 5 5 19 20 21 22 This study has indicated, for the first time that the RANKL-dependent pathway is important in the pathogenesis of Charcot’s osteoarthropathy, thereby raising the possibility of the use of RANKL inhibition in the management of Charcot’s foot. However, our observations also suggest that a RANKL-independent pathway may play a role, but further investigation is required to fully clarify the mechanism involved. If confirmed, specific pharmacological agents that counteract the RANKL-independent pathway, such as anti-TNF strategies, may be useful in the treatment of Charcot’s osteoarthropathy. Whatever the relative importance of either pathway, this in vitro technique of generating human osteoclasts from PBMCs may allow specific characterisation of osteoclastic activity in each patient and could, in the future, lead to individually tailored anti-osteoclastic treatment for the patient with acute Charcot’s osteoarthropathy.