IADR Abstract Archives
Regulation of osteoclasts’ shedding of RANK-containing extracellular vesicles
Objectives: Proper coupling of bone resorption by osteoclasts with bone formation by osteoblasts is crucial for healthy bone. Extracellular vesicles containing receptor activator of nuclear factor kappa B (RANK-EVs), which are shed by osteoclasts, were recently identified as coupling regulators in bone remodeling. We recently reported that osteoclasts resorbing bone in cell culture shed more RANK-EVs than when resorbing dentine. Here, we have sought other factors that regulate the shedding of RANK-EVs by osteoclasts.
Methods: Primary osteoclasts from mouse marrow and RAW 264.7 osteoclast-like cells were cultured by standard methods. Bone biomimetics were generated by the polymer-induced liquid precursor process using osteopontin to produce aligned nanocrystals of hydroxyapatite in the interstices of collagen fibrils. Cells were stretched at 3% elongation using a sinusoidal strain application form at a frequency of 0.5 Hz for 6-hours per day in a Flexcell system. Osteoclasts were identified by staining for tartrate-resistant acid phosphatase activity. Activated osteoclasts were detected by staining actin rings with rhodamine-phalloidin. EVs were isolated by ExoQuick, followed by ultracentrifugation. EV proteins were then analyzed by SDS-PAGE and immunoblots and RANK was detected using an anti-RANK antibody (Biorbyt).
Results: Osteoclasts resorbing bone biomimetics shed fewer RANK-EVs compared with bone, though osteoclast activation was similar. Osteoclasts on material extracted from bone with EDTA, then dried on plastic, stimulated increased shedding of RANK-EVs. Osteoclast-like cells subjected to a stretching regimen produced more RANK-EVs at 2-days of stretching than unstretched controls, and similar numbers at 5 days. Osteoclasts cultured on fibronectin produced fewer RANK-EVs than controls though the number of osteoclasts produced by the cultures was not significantly different.
Conclusions: Various conditions that regulate RANK-EV release by osteoclasts were identified. These provide model systems for studying the underlying molecular mechanisms that regulate shedding of RANK-EVs by osteoclasts.
Methods: Primary osteoclasts from mouse marrow and RAW 264.7 osteoclast-like cells were cultured by standard methods. Bone biomimetics were generated by the polymer-induced liquid precursor process using osteopontin to produce aligned nanocrystals of hydroxyapatite in the interstices of collagen fibrils. Cells were stretched at 3% elongation using a sinusoidal strain application form at a frequency of 0.5 Hz for 6-hours per day in a Flexcell system. Osteoclasts were identified by staining for tartrate-resistant acid phosphatase activity. Activated osteoclasts were detected by staining actin rings with rhodamine-phalloidin. EVs were isolated by ExoQuick, followed by ultracentrifugation. EV proteins were then analyzed by SDS-PAGE and immunoblots and RANK was detected using an anti-RANK antibody (Biorbyt).
Results: Osteoclasts resorbing bone biomimetics shed fewer RANK-EVs compared with bone, though osteoclast activation was similar. Osteoclasts on material extracted from bone with EDTA, then dried on plastic, stimulated increased shedding of RANK-EVs. Osteoclast-like cells subjected to a stretching regimen produced more RANK-EVs at 2-days of stretching than unstretched controls, and similar numbers at 5 days. Osteoclasts cultured on fibronectin produced fewer RANK-EVs than controls though the number of osteoclasts produced by the cultures was not significantly different.
Conclusions: Various conditions that regulate RANK-EV release by osteoclasts were identified. These provide model systems for studying the underlying molecular mechanisms that regulate shedding of RANK-EVs by osteoclasts.