Histomorphometry

All bone-specific parameters will be measured and expressed in units following the guidelines established by the ASBMR histomorphometry nomenclature committee (5). For each experiment both experimental and control samples will be analyzed simultaneously, with a minimum of 15 animals per group to minimize the influence of genetic variability.

Initially, only the four sections per specimen treated by the von Kossa reagent (that stains black mineralized bone matrix) will be analyzed. For vertebrae both L3 and L4 will be analyzed. Using the semi-automatic mode existing in the Osteomeasure system, we will measure three parameters:

• Bone volume

Calculated as the total bone marrow volume occupied by trabecular bone (BV/TV, %)

• Trabecular number

Evaluated as the number of trabeculae present in the field analyzed for the bone volume (Tb.N, mm^-1)

• Trabecular thickness

Calculated as the averaged thickness of the trabeculae present in the field analyzed for the bone volume (Tb.Th, µm)

These parameters will provide a reliable estimate of the existence of an increased or decreased bone mass in experimental specimens. Whether there is such a phenotype and the necessity of additional analyses will be evaluated. Cell counts and dynamic histomorphometry require processing additional sections for specific stainings and/or are extremely time-consuming. These additional analyses will only be performed upon request by the project investigators.

Cell counts and dynamic histomorphometry will be performed on 30-35 adjacent fields of high magnification images obtained from two non-adjacent sections.

Measurements will be as follows:

Osteoblasts will be counted on toluidine blue stained slides as blue/grey cuboïdal cells aligned in clusters at the bone surface. Both osteoblast number (N.Ob/BPm, mm^-1) and osteoblast surface (Ob. S/BS, %) data will be provided to investigators.

Osteoclasts will be counted on slides assayed for TRAP activity as TRAP-positive multinucleated cells. Both osteoclast number (N.Oc/BPm, mm^-1) and osteoclast surface (Oc.S/BS, %) data will be provided to investigators.

Two dynamic parameters characterizing bone formation will be provided to investigators. The mineral apposition rate (MAR, mm/day) will be measured as the distance between the midpoints of the two consecutive calcein labels divided by the time interval between the labeling periods. The bone formation rate (BFR/BS, mm³/mm²/day) will be calculated as an expression of the amount of newly formed mineralized bone.