Bone fragility can be caused by low bone mass or by alterations in bone material composition. While mineral makes bone strong, hypermineralisation makes bone brittle. We have reported that hypermineralisation can be associated with lysosome deficiency in osteocytes. Lysosomes are intracellular acidic vesicles containing degradative enzymes, including cysteine proteases (CPs, e.g. cathepsins B, L and X). We therefore hypothesized that osteocytes use CPs to limit mineral accumulation in bone.
When Ocy454 osteocyte-like cells were treated with the broad-spectrum CP inhibitor E-64, we observed a significant, dose-dependent increase in mineral accumulation using Alizarin Red Stain in vitro.
In vivo, cathepsin activity was measured 1 hour after single injections of vehicle or E-64 (5, 10 or 50mg/kg)) above the calvaria in six-week-old female C57/BL6 mice. Using the BMV109 probe which, upon cathepsin-mediated cleavage, binds to the active cathepsins and emits a fluorescent signal, we found that 10mg/kg was sufficient to lower cathepsin B, L and X activity.
Therefore, six-week-old female C57/BL6 mice received first oncostatin M (0.2µg, daily for five days) to promote new bone formation, then E-64 (10mg/kg, daily for five days) over the calvaria. Micro-computed tomography of the non-remodelling bone surface showed a significantly (~5%) greater tissue mineral density in calvariae of E-64-treated mice versus controls, with no difference in cortical thickness. Fourier transform infrared microspectroscopy revealed significantly greater mineral:matrix and carbonate:mineral ratios in E-64-treated bones compared to controls. This was restricted to older bone, located 1.2mm posterior to the sagittal suture. Both results indicate that CP inhibition leads to greater mineral content and carbonate substitution.
Overall, E-64 treatment led to greater mineral accrual and higher mineral maturity without changing bone mass. This suggests that osteocytes use cysteine proteases to maintain bone material quality and highlights their therapeutical potential to change bone material composition within existing bone.