A Sectioning, Coring, and Image Processing Guide for High-Throughput Cortical Bone Sample Procurement and Analysis for Synchrotron Micro-CT

Available data related to bone specimen procurement for synchrotron micro-computed tomography remain sparse. Our comprehensive tutorial provides a methodology that is straightforward, minimally destructive, and cost-effective. Procuring bone specimens with consistent dimensions and a cylindrical shape is critical to ensure that the resulting data sets are of the highest quality and the results are applicable.

The techniques described in this manuscript are applicable to coring rocks, fossils, or any hard material. We've used these to collect uniformly sized cores of rocks and single crystals for high-pressure experiments to explore the rheology of Earth's crust and upper mantle. Novice corers may find that the bone specimen forms conical shapes.

This can be addressed by allowing adequate and enough time for bone dust to flush from the drill bit and slowing down coring speed. There's a lack of instructional texts and videos that demonstrate bone specimen procurement for synchrotron micro-computed tomography and is the logical analysis. Our step-by-step bone preparation tutorial helps to fill this out gap.

Place a 75 by 25 millimeter glass microscope slide on hot plates up to 140 degrees Celsius and melt a generous amount of thermal epoxy resin on the center of the slide. Press the inferior aspect of the bone block into the thermal epoxy resin on the microscope slide with the length of the bone perpendicular to the slide. Shift the sample back and forth in order to coat the underside of the bone and ensure secure adhesion.

Let the mounted specimen rest on the hot plate for approximately five minutes to allow the thermal epoxy to wick into the pores and cracks making sure that the epoxy on the slide is free of bubbles. If bubbles are present, shift the sample back and forth to remove them. Use blunt forceps to remove the slide with the mounted specimen from the hot plate and allow it to cool at room temperature for about 10 minutes, then remove any epoxy from the edge of the slide with a razor blade to ensure that the chuck adequately grips the slide.

Attach the slide with the adhered sample to a glass slide chuck and mount the chuck on the swivel arm of a slow speed sectioning saw positioning it so that a cross-section of the bone can be cut perpendicular to its length. Adjust the swivel arm to ensure that the blade contacts and transects the sample. Add weights to the far side of the cutting arm to counter the weight of the arm and add distilled water and cutting fluid to the fluid receptacle.

Tightly secure the diamond wafer blade and ensure that the fluid level submerges the cutting portion of the blade. Set the speed to 200 RPM and slowly lower the sample onto the blade. Once the saw begins sectioning, ensure that the blade and chuck are not wobbling or bouncing.

If they are, immediately stop the saw and tighten the blade or chuck arm assembly. If the chuck is aggressively moving up and down, add more counterweights. The first thick section is a waste cut that will provide a well-defined surface parallel to each additional cut.

After the initial waste cut, raise the swivel arm and move the chuck five millimeters towards the blade using the positioning dial. After sectioning is complete, place the glass slide with the mounted specimen on a hot plate to melt the thermal epoxy. Mount five millimeter bone sections to the bottom of a shallow aluminum tin using the thermal epoxy bonding technique previously described.

Place the tin on an XY machine table of the mill drill press and hand-tighten the fixturing clamps. Insert a two millimeter inner diameter hollow shaft jewelers diamond tipped coring drill bit into the mill drill chuck and adjust the depth limiter to prevent coring through the tin. Align the central anterior aspect of the bone sample beneath the drill bit while avoiding close contact with the periosteum and ostium or highly trabecularized areas.

Fill the tin with distilled water to completely cover the sample which prevents heat buildup, burning of the sample, and damage to the drill bit during coring. The mill drill press can be dangerous if proper safety measures aren't taken. Operators should ensure that they wear safety glasses, that loose clothing is secured, and that long hair is pulled back so it doesn't get caught in the spindle.

for the first few instances of contact between the core bit and bone, apply gentle pressure in order to wear a ring on the superior surface of the bone. This prevents deflection of the drill bit at the beginning of the coring process and ensures correct placement of the bit. During coring, lift the drill bit in and out of the sample while keeping the bit's tip beneath the water surface.

Do this every few seconds to flush out trapped bone dust and ensure debris is not occluding the drill bit. After coring is complete, the resulting bone core may become lodged in the hollow stemmed drill bit. Use a pair of fine tipped forceps or a small Allen wrench to dislodge the core from the bit.

Store the cored sample in a labeled microcentrifuge tube in a cool and dry location until imaging. The described method of core sampling proved to be highly effective and efficient. Representative figures comparing the image processing workflow of a cored sample and one procured using a rotary tool are shown here.

The sample cut using the common rotary tool exhibited an increased number of canals and lacunae and a decreased average canal diameter, canal volume, and cortical porosity when compared to the cored sample. This table shows porosity data for each sample. Although the coring protocol decreases artifacts observed in synchrotron microcomputer tomography scans, the lower quality artifact-laden figures from the rectilinear bone block experiments represent a multifaceted issue.

Subsequent image processing confirmed the potential of the technique to improve visualization of cortical bone micro architecture. For example, mineralization differences, improved delineation of osteonal boundaries, and consistent visualization of soft tissues within vascular canals were observed. Proceeding slowly is the key to getting consistently-sized cylindrical samples.

Going too fast can cause the specimens to become cone-shaped instead of cylindrical. While procuring thick sections for coring, further thick sections can be gathered for Brightfield or confocal microscopy. This allows for the visualization of the canalicular network.