Cortical Bone Protocol
Sectioning & Imaging
Cross-sections of the diaphysis are usually prepared using a grinding system rather than a microtome. Attempting to cut a cross-section of the diaphysis with a microtome usually causes the section to disintegrate even when resin-embeded. Typically, a 100 micron slab is cut with a saw then mounted on a plastic slide. Then slab is then ground and polished down to a thickness of about 30 microns.
Sections may be given a light surface stain. Sections taken from mice are normally imaged with a 20X objective. Section from large animals such as mini-pigs are normally imaged with a 10X objective.
Imaging of the section is generally done in bright field. Fluorescence may be used in cases where labels have been given to the animals as can be the case in studies that use 3-point bending to look at response to mechanical loading. Polarized light may sometimes be used to help visualize the borders of osteons tunneled through lamellar bone.
Whole Section Referent Data
cortical area, thickness, & periosteal surfacE
Using the digital scan of the section, BIOQUANT zooms out so the entire cross-section is visible within the field of view. Based on a threshold of the cortical bone, BIOQUANT collects the cortical area, the cortical thickness, and the periosteal surface.
- Mean Ct.Wi
2nd moment of AREA
Relative to the X axis of the image, BIOQUANT also computes the 2nd moment of area for the cross-section. This is a measure of the distribution of cortical bone in section. To be comparable to one another, sections must be images in a consistent anatomical orientation.
Anterior / Posterior / Medial / Lateral Zone Data
Cortical Thickness along Anatomical Axes
The software provides tools for the direct measurement of cortical thickness at preferred anatomical orientations. Typically, four thickness measurements are made: anterior, posterior, media, and later.
- Anterior Ct.Wi
- Posterior Ct.Wi
- Medial Ct.Wi
- Lateral Ct.Wi
Sampling Along Anatomical Axes
Sampling Areas at the four orientations are defined using a standardized Region of Interest tool that allows strips of standardized shape to be analyzed at higher magnification.
- Sa.Ct.Ar Sampled Cortical Area
Osteon Area / Osteon Perimeter
Since there is no specific staining at the border of an osteon, the boundary is drawn by hand. Once, drawn the boundary is analyzed for both area and perimeter.
- On.Ar Osteonal Area
- On.Pm Osteonal Perimeter
- On.N Osteon Number
- OPD Osteon Population Density
- Ha.Ar Haversian Canal Area
- Ha.Pm Haversian Perimeter
- On.Fg.Ar Osteon Fragment Area
- On.Fg.N Osteon Fragment Number
- Po.Ar Pore Area
Osteon Diameters & Wall Thickness
Similarly, the major and minor diameters of the osteon as well as the wall thickness along the minor diameter of the osteon are manual measurements.
- Min.On.Dm Minimum Osteon Diameter
- Max. On.Dm Maximum Osteon Diameter
- W.Wi Osteon Wall Width
BIOQUANT automatically counts osteocytes using color, size, and shape to identify them. The Measure Cells tool ensures that no osteocyte is counted twice and that only osteocytes within the sampling area are counted.
- Ot.N Osteocyte Number
Bone Formation Rate Data
Auto Surface tool for single labeled surfaces
Using the Auto Surface tool, BIOQUANT automatically measures the single labeled surface. Manual editing of the labeled surface threshold is easily done with the Draw and Erase tools.
- sL.Pm Single Labeled Surface
Specialized Double Labeled Surface tool
Pairs of labeled surfaces are traced manually. The dLS tool then automatically measures the interlabel width and the length of the double labeled surface. This data is combined with single labeled surface and bone surface to automatically calculate
- dL.Pm Double Labeled Surfae
- M.Pm Mineralizing Perimeter