Histo terms

Definition of Terms: Histomorphometry

Region of interest (ROI).

This is the area in which the computer measurements will be made. The mineralized morphometry of the bone is defined by fusing the darkfield and calcein blue signals that are captured during the first round of imaging. The ROI is selected by the computer algorithms for each tissue that is imaged and is applied to all the subsequent staining and imaging steps.

Trabecular bone ROI:

The region of interest (ROI) is determined from this image as a fixed area (2.15 mm2) that is 400µm beneath the growth plate and 200-300 µm internal from the endocortical bone surface. This rule can cause problems because of the width of the metaphysis varies significantly between male and females, as well as between different KO lines. Thus mice with a narrow metaphysis will acquire a sample of that extends into area of diaphyseal bone where the trabecular density can become very low. This step may falsely lower the calculated BV/TV measurement. The analysis performed by the µCT community does not follow this rule but instead controls the distance away from the growth plate as a function of the overall bone length to prevent sampling from the diaphyseal region. Currently we are following the same ROI selection rules as the classical histomorphometry.

Cortical bone ROI:

The cortical bone adjacent to the ROI defined for the femoral trabecular bone is used as region of analysis. Both sides are queried and the results are combined

Vertebral body ROI:

In the body of the vertebra, it is ?50µ from the cortex and does not extend into the ? that extend out from the vertebral body.

Static histomorphometry of the trabecular bone (femur and vertebra):

This measurement assesses the architectural properties of the bone. The µCT provides a more comprehensive and reproducible assessment of bone architecture because the entire volume of the bone is assessed. However the step is performed by histology as the mineralized template to map the dynamic measurements to bone surfaces (BS) as well as a confirmatory measurement for µCT. T here is reasonable correlation between the static measurements obtained by µCT and histology (figure x).

In the tables that follow, the direct measurements are preceded by an x, while calculation based on direct measurements are preceded by a c.

Abbreviation

Name

Units

Type of measure

BV/TV

Bone volume per total volume%

Calculated

The percentage of area within the ROI (TV) that emits the signal indicating accumulated mineral (BV). It is a planar measurement in histology (the mean of 3 different levels) in contrast to the volumetric value of the entire ROI in µCT. Similar to µCT, the males have a greater BV than females in the femur but the difference between the two genders is much less in the vertebra. Consistent with the µCT data, there is greater variation in BV/TV measurements in male versus the female group.

Tb.Th.

Trabecular thickness

µm

Direct

It is the average thickness (not length) of each bony element within the ROI. The computer recognizes the trabecular surface by the presence of the surface labeling signals and measures the thickness directly.

Tb.N.

Trabecular Number

#/mm

Calculated

The measurement is the average number of trabeculae encounter by any arbitrary line drawn within ROI. It is a calculation of the (BV/TV)/Tb.Th. The number is independent of trabecular size.

Tb.SP.

Trabecular spacing

Calculated

It is the average space length of any arbitrary line drawn within ROI. It is a calculation of the (1/Tb.N. – Tb.Th.).

Static histomorphometry of the cortical bone:

This µCT study provides many more and better measures of the cortical bone because it assesses the entire volume of the sample. The histological measurement of cortical thickness is affected by the angle of sectioning. However, the histology can estimate osteocyte density which should be independent of the plane of section and it is a measurement that the µCT cannot provide.

Abbreviation

Name

Units

Type of measure

C.B.Th

Cortical bone thickness

µM

CDirect

The average thickness of the cortical bone adjacent to the ROI expressed in µ. It is not a reliable measurement due to plane of section effect.

Oc.N.

Osteocyte density

# per µ2

Direct

IThe number of DAPI positive cells restricted to the cortical bone. This measurement is obtained from staining step 3 (AP activity) that utilizes DAPI as a nuclear stain. The value cannot be made with conventional µCT instruments.

H/W

Height to width ratio of the distal femur

Ratio, no units

Direct

A crude assessment of the shape of the femoral bone. It is calculated as the ratio of the fixed size ROI rectangle. The greater the ratio, the narrower the metaphyseal bone and the further ROI extends into the diaphyseal bone.

Definition of Terms: Histomorphometry
Region of interest (ROI). This is the area in which the computer measurements will be made. The mineralized morphometry of the bone is defined by fusing the darkfield and calcein blue signals that are captured during the first round of imaging. The ROI is selected by the computer algorithms for each tissue that is imaged and is applied to all the subsequent staining and imaging steps.
Trabecular bone ROI: The region of interest (ROI) is determined from this image as a fixed area (2.15 mm2) that is 400µm beneath the growth plate and 200-300 µm internal from the endocortical bone surface. This rule can cause problems because of the width of the metaphysis varies significantly between male and females, as well as between different KO lines. Thus mice with a narrow metaphysis will acquire a sample of that extends into area of diaphyseal bone where the trabecular density can become very low. This step may falsely lower the calculated BV/TV measurement. The analysis performed by the µCT community does not follow this rule but instead controls the distance away from the growth plate as a function of the overall bone length to prevent sampling from the diaphyseal region. Currently we are following the same ROI selection rules as the classical histomorphometry. Cortical bone ROI: The cortical bone adjacent to the ROI defined for the femoral trabecular bone is used as region of analysis. Both sides are queried and the results are combined Vertebral body ROI: In the body of the vertebra, it is ?50µ from the cortex and does not extend into the ? that extend out from the vertebral body.
Static histomorphometry of the trabecular bone (femur and vertebra): This measurement assesses the architectural properties of the bone. The µCT provides a more comprehensive and reproducible assessment of bone architecture because the entire volume of the bone is assessed. However the step is performed by histology as the mineralized template to map the dynamic measurements to bone surfaces (BS) as well as a confirmatory measurement for µCT. T here is reasonable correlation between the static measurements obtained by µCT and histology (figure x). In the tables that follow, the direct measurements are preceded by an x, while calculation based on direct measurements are preceded by a c.
Abbreviation	Name	Units	Type of measure
BV/TV	Bone volume per total volume%	%	Calculated
BV/TV	The percentage of area within the ROI (TV) that emits the signal indicating accumulated mineral (BV). It is a planar measurement in histology (the mean of 3 different levels) in contrast to the volumetric value of the entire ROI in µCT. Similar to µCT, the males have a greater BV than females in the femur but the difference between the two genders is much less in the vertebra. Consistent with the µCT data, there is greater variation in BV/TV measurements in male versus the female group.
Tb.Th.	Trabecular thickness	µm	Direct
Tb.Th.	It is the average thickness (not length) of each bony element within the ROI. The computer recognizes the trabecular surface by the presence of the surface labeling signals and measures the thickness directly.
Tb.N.	Trabecular Number	#/mm	Calculated
Tb.N.	The measurement is the average number of trabeculae encounter by any arbitrary line drawn within ROI. It is a calculation of the (BV/TV)/Tb.Th. The number is independent of trabecular size.
Tb.SP.	Trabecular spacing	mm	Calculated
Tb.SP.	It is the average space length of any arbitrary line drawn within ROI. It is a calculation of the (1/Tb.N. – Tb.Th.).
Static histomorphometry of the cortical bone: This µCT study provides many more and better measures of the cortical bone because it assesses the entire volume of the sample. The histological measurement of cortical thickness is affected by the angle of sectioning. However, the histology can estimate osteocyte density which should be independent of the plane of section and it is a measurement that the µCT cannot provide.
Abbreviation	Name	Units	Type of measure
C.B.Th	Cortical bone thickness	µM	CDirect
C.B.Th	The average thickness of the cortical bone adjacent to the ROI expressed in µ. It is not a reliable measurement due to plane of section effect.
Oc.N.	Osteocyte density	# per µ2	Direct
Oc.N.	IThe number of DAPI positive cells restricted to the cortical bone. This measurement is obtained from staining step 3 (AP activity) that utilizes DAPI as a nuclear stain. The value cannot be made with conventional µCT instruments.
H/W	Height to width ratio of the distal femur	Ratio, no units	Direct
H/W	A crude assessment of the shape of the femoral bone. It is calculated as the ratio of the fixed size ROI rectangle. The greater the ratio, the narrower the metaphyseal bone and the further ROI extends into the diaphyseal bone.