Skeletons Come To Light
To strengthen the skeleton, bone-gnawing cells called osteoclasts must first actually break it down. To catch osteoclasts in action, researchers have now developed fluorescent probes that stick to bone and light up only when osteoclasts are chomping on bone nearby (J. Am. Chem. Soc., DOI: 10.1021/ja2064582). Spying on osteoclasts could help scientists develop new treatments for bone diseases like osteoporosis.
Osteoclasts dwell in pits on the surfaces of bones and release protons, causing the pH in the pit to drop. Acidification helps the osteoclast remove bone tissue in a process called resorption. Sometimes, though, osteoclasts resorb too much or too little, sabotaging the structural integrity of the bone. “To date, almost all the drugs used for bone diseases target the function of osteoclasts,” says Masaru Ishii of Osaka University.
Currently, doctors and scientists measure osteoclast behavior indirectly, including by measuring biomarkers of bone resorption. But Ishii wants to know the details of bone remodeling, including when and where it happens, and why it sometimes goes wrong. So he and his colleagues devised a way to catch osteoclasts in the act of dissolving bone. They made a chemical probe containing a fluorophore, a green dye called boron dipyrromethene, that is sensitive to pH. They linked the dye to bisphosphonate, a compound with a natural affinity for bone. The researchers made three probes, two that fluoresce at acidic pHs and a control that’s perpetually on.
To test the probes, the researchers mixed them with pieces of hydroxyapatite, a major component of bone, in buffers with different pHs. They detected fluorescence with confocal microscopy. When mixed with the indiscriminate probe, hydroxyapatite glowed green regardless of buffer conditions. In the presence of the pickier probes, it only lit up in acidic buffers.
Next, the researchers injected the probes daily for three days into transgenic mice, chosen because their osteoclasts contain a red fluorophore. Then, the researchers surgically exposed the top of the rodents’ skulls. To measure fluorescence, they used two-photon microscopy, which penetrates deep inside bone tissue. The bones of mice injected with the always-on probe glowed green all over, while those of mice with the pH-sensitive probes contained just a few green spots, showing acidic regions. These spots, Ishii says, revealed exactly where the osteoclasts were resorbing bone. The researchers confirmed this interpretation by observing red-glowing osteoclasts in the vicinity of the green spots.