Gauchers disease is a glycolipid storage disorder caused by an autosomally inherited deficiency of the lysosomal enzyme glucocerebrosidase. The majority of patients with Gauchers disease develop abnormal bone remodelling with severe consequences, including osteonecrosis, bone crises, and osteoporosis related fractures. Although enzyme replacement therapy is effective at reversing many of the pathological consequences, and preventing further progression of this disease, only modest improvements in bone health can be achieved. The reason for this apparent bone resistance is unclear but may reflect abnormal osteoclast function. To address this problem, we developed an in vitro model of osteoclast bone resorption, in which we differentiated mouse RAW264.7 cells, into functional osteoclasts using the cytokine RANKL. Multinucleated TRAP (tartrate resistant alkaline phosphatase) positive cells were apparent after 7 days of culture on uncoated tissue culture plastic.
Osteoclasts were differentiated on inorganic calcium phosphate coated plates designed to mimic the in vivo bone environment. Cultures were maintained for 14 days and the Gauchers defect was modelled by culturing cells in the presence of conduritol β epoxide a potent, irreversible inhibitor of mammalian glucocerebrosidase. After the culture period, cells were removed and the number of resorption pits counted. We observed that the number of osteoclasts and resorption pits was increased by 20% following culture in the presence of 50 μM CBE and this effectively doubled at 100 μM.
Our novel in vitro model of Gauchers osteoclasts recapitulates observations made in studies using osteoclasts derived from the peripheral blood of patients with Gauchers disease. This system may provide a useful tool for understanding the mechanisms of increased bone turnover in Gauchers disease and in particular the intracellular consequences of defective glucocerebrosidase activity within the osteoclast. This may provide insights for the development of alternative or complementary therapies to prevent the aberrant bone turnover associated with this rare disease.
17 May 2014 - 20 May 2014