Two types of bone specimen will be analyzed by the Core: Skeleton from genetically modified animals and long bones injected with prostate cancer cells. In the former case both vertebrae (lumbar vertebrae L2, L3 and L4) and tibiae will be embedded, but only L3 and L4 vertebrae will be initially analyzed. Sectioning and analysis of the tibiae will be performed only at special request and after consultation between the Core personnel and the principal investigator of the project. For tumor cell injected mice, both the injected bone and its non-injected counterpart will be embedded and analyzed.
Processing/Embedding/Staining of Non-Decalcified Bone Specimen
Calcein-labeled (1) skeletons fixed flat in 10% formalin for no more than six hours at 4°C and dehydrated up to 80% ethanol will be provided to the core by project investigators. According to the type of experimental model (bones of genetically modified mice or injected bones), appropriate specimen will be dissected and dehydrated at 4°C in alcoholic solutions up to 100% ethanol. The remainder of the skeleton will be sent back to investigators. Dissected specimen will be processed according to established protocols (2). They will be infiltrated twice 24 hours at 4°C in a media containing 100 ml destabilized methylmethacrylate (MMA), 14 ml nonylphenyl-polyethyleneglycol acetate (NPG) and 0.33 g anhydrous benzoyl peroxide (BPO). Samples will then be transferred in a medium containing 100 ml MMA, 14 ml NPG, 0.55 g BPO and 500 ml N,N dimethyl-p-toluidine 99%. Polymerization will be carried out at 4°C.
Plastic blocks will be trimmed on a grainer/polisher and sectioning will be performed on a motorized Leica RM2165 microtome equipped with tungsten-carbide disposable blades. Particular attention will be given in positioning the blocks to insure that all specimens have the same orientation when sectioned. Seven micrometers sections will be collected throughout the bones of injected mice (sagittal sections) or until the two third of the vertebrae are reached for genetically modified animals or their control littermates. A set of 10 consecutive sections, located in the same plane between animals, will be selected (#1-10) for analysis. Sections #1-3 and 8-10 (7 microns thick) will be stained with the von Kossa reagent and counterstained by the van Gieson method for initial analysis of the basic parameters (3). This procedure stains mineralized bone matrix in black, osteoid in red and the cells medium pink. Sections 4-7 will be cut at four microns thickness and left unstained and stored for further analyses. Upon particular request, and on a case-by-case basis, these unstained sections will be used to perform cell counts (osteoblasts, osteoclasts) and quantify dynamic parameters (BFR).
For quantification of osteoblast numbers, slide # 4 will be stained with toluidine blue according to a standard protocol (4). For quantification of osteoclast numbers slides # 5 will be subjected to an enzymatic assay for detection of tartrate resistant alkaline phosphatase staining (TRAP) (4), an enzyme specifically expressed by osteoclasts in the bone marrow. The sections will then be stained with faded hematoxylin. Slide # 6 will be left unstained for visualization of calcein at the mineralization fronts under fluorescent light. Measurements will be performed in 30-35 fields on one slide per sample. All slides within a sample will be examined for possible abnormalities which are avoided.
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 concurrently. It is recommended to analyze a minimum of 15 animals per group to lessen the influence of possible genetic variability.
Analyzing either the long bones (i.e. femur, tibia) or skeletal bone (lower spine -specifically L3 or L4- bone biopsy, or calvarium), we will use the semi-automatic mode existing within the Osteomeasure system to measure three parameters:
- Bone volume
- Calculated as the total bone marrow volume occupied by trabecular bone (BV/TV, %); bone volume should be greater than or equal to 15% of measurement area. Analysis may require more than one bone section to achieve adequate bone volume
- Trabecular number
- Evaluated as the number of trabeculae present within the field of measure for bone volume (Tb.N, mm-1)
- Trabecular thickness
- Trabecular thickness
- Calculated as the average thickness of the trabeculae present within the field of measure of the bone volume (Tb.Th, µm)
These parameters will provide a reliable estimate of the existence of the increased or decreased bone mass in experimental specimens.
Cell counts and dynamic histomorphometry will be performed on 30-35 adjacent fields of high magnification images obtained from two non-adjacent sections/slides.
Cell Type and Measurement Processes
Osteoblasts are cuboidal cells aligned in clusters along the bone surface. Osteoclasts are large, tartrate-resistant acid phosphatase (TRAP)-positive, multinucleated cells along the bone surface Osteoblast/Osteoclast cell count and dynamic histomorphometry require specific staining for evaluation and can be time-consuming. These services can be performed upon request by the project investigators.
Osteoblasts can be best visualized using either the Toluidine Blue, HTX or Goldner’s Modified Trichrome stain. Both osteoblast number (N.Oc/BPm, mm-1) and osteoblast surface (Ob.S/BS, %) data will be provided to investigators.
Osteoclasts can be best visualized using a TRAP enzymatic stain. Both osteoclast number (N.Oc/BPm, mm-1) and osteoclast surface (Oc.S/BS, %) data will be provided to investigators.
Data Investigator Receives
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 two distinct calcein labels administered by injection divided by the time interval between the labeling events. Mineralizing surface (MS) is calculated by adding double-label surface and one half the single-label surface and divided by the total bone surface. The bone formation rate (BFR/BS, mm3/mm2/day) is calculated by multiplying the MAR by the MS.
Time intervals are defined as follows for calcein injections:
- 10-14 days in large animals
- 2-7 days in small animals; a 4-day interval have proven ideal for small rodents