Scientists devise a technique to make bones transparent, enabling them to observe the stem cells inside

A mouse tibia that has been rendered transparent with Bone CLARITY. Stem cells appear distributed throughout the bone in red. The ability to see bone stem cell behavior is crucial for testing new osteoporosis treatments.
Credit: Science Translational Medicine, Greenbaum, Chan, et al; Gradinaru laboratory/Caltech

What if you could see through the opaque bones and get an accurate picture of the bone’s internal structure? It would be an incredibly useful tool for research into bone diseases.

Caltech neuroscientist Viviana Gradinaru who is also the Assistant professor of biology and biological engineering at the Heritage Medical Research Institute has devised a chemical technique which could make the mouse bones transparent! Though such techniques have been used in the past to make see-through kidneys and brains, this is the first time it has been attempted on hard tissues like bones.

Our bones are not static organs, we don’t have the same bones we had 10 years back. In a healthy bone, there exists a delicate balance between the cells that build bone mass and the cells that break down old bone, in a continual remodeling cycle.

This process of growth and death in the bone cell mass is regulated by stem cells in the bone marrow called osteoprogenitors. To better understand diseases like osteoporosis, which occurs when loss of bone mass leads to a high risk of fractures, it is crucial to study the behavior of stem cells in bone marrow. However, this population is rare and not distributed uniformly throughout the bone.

“Because of the sparsity of the stem cell population in the bone, it is challenging to extrapolate their numbers and positions from just a few slices of bone,” says Alon Greenbaum, postdoctoral scholar in biology and biological engineering and co-first author on the paper. “Additionally, slicing into bone causes deterioration and loses the complex and three-dimensional environment of the stem cell inside the bone. So there is a need to see inside intact tissue.”

Using a technique called CLARITY, originally developed for clearing brain tissue during Gradinaru’s postgraduate work at Stanford University, the team set out to visualize the bones in 3D. They developed a way to clear hard tissues, like the bone that makes up our skeleton.

CLARITY renders soft tissues, such as brain, transparent by removing opaque molecules called lipids from cells while also providing structural support by an infusion of a clear hydrogel mesh.

How did they do it? 

The team began with bones taken from postmortem mice, which was genetically altered to have their stem cells fluoresce red, so that they could be easily imaged. They chose the femur and tibia as samples to analyze. Initial treatment involved removal of calcium from these bones as it contributes to the opacity. Next, because lipids also provide tissues with structure, the team infused the bone with a hydrogel that locked cellular components like proteins and nucleic acids into place and preserved the architecture of the samples.

This was followed by a gentle detergent treatment (EDTA, SDS, amino alcohol) to remove the fats that made the bone transparent to the eye. What remains at the end of this treatment is an intact bone that was almost completely, transparent.

For imaging the cleared bones, the team built a custom light- sheet microscope for fast and high-resolution visualization that would not damage the fluorescent signal. The cleared bones revealed a constellation of red fluorescing stem cells inside.

Practical Applications 

The group collaborated with researchers at the biotechnology company Amgen to use the method, named Bone CLARITY, to test a new drug developed for treating osteoporosis, which affects millions of Americans per year.

“Our collaborators at Amgen sent us a new therapeutic that increases bone mass,” says Ken Chan, graduate student and co-first author of the paper. “However, the effect of these therapeutics on the stem cell population was unclear. We reasoned that they might be increasing the proliferation of stem cells.”

To test this, the researchers gave one group of mice the treatment and, using Bone CLARITY, compared their vertebral columns with bones from a control group of animals that did not get the drug.

“We saw that indeed there was an increase in stem cells with this drug,” he says. “Monitoring stem cell responses to these kinds of drugs is crucial because early increases in proliferation are expected while new bone is being built, but long-term proliferation can lead to cancer.”

The technique has promising applications for understanding how bones interact with the rest of the body.

“Biologists are beginning to discover that bones are not just structural supports,” says Gradinaru, who also serves as the director of the Center for Molecular and Cellular Neuroscience at the Tianqiao and Chrissy Chen Institute for Neuroscience at Caltech. “For example, hormones from bone send the brain signals to regulate appetite, and studying the interface between the skull and the brain is a vital part of neuroscience. It is our hope that Bone CLARITY will help break new ground in understanding the inner workings of these important organs.”

The paper is titled “Bone CLARITY: Clearing, imaging, and computational analysis of osteoprogenitors within intact bone marrow.”