Osteomeasure

To detect and quantify bone formation, calcein double labels were assessed in fluorescent light microscopy. The following parameters were evaluated using OsteoMeasure (OsteoMetrics, Atlanta, GA, USA): The mineralizing surface per bone surface (MS/BS,%), representing active mineralization, calculated as percent of bone surface that displays a calcein label; the mineral apposition rate (MAR, µm/d), representing the rate of new bone deposition, calculated as distance between consecutively deposited double labels divided by the time interval between them; and the bone formation rate (BFR, µm³/µm²/y), representing the amount of new bone formed, calculated by multiplying mineralizing surface with the mineral apposition rate.
Osteoblast- and osteoid-related parameters were also quantified using the OsteoMeasure histomorphometry system. The parameters - number of osteoblasts per bone perimeter (N.Ob/B.Pr, 1/mm), osteoblast surface per bone surface (Ob.S/BS, %) as well as osteoid indices, including osteoid surface per bone surface (OS/BS, %) and osteoid thickness (O.Th, µm), were extracted in order to quantify cell based bone deposition activity. In each sample section, eight microscopic fields were evaluated at 20-fold magnification. The cells were counted in Masson-Goldner-stained sections, and osteoid indices where evaluated in von Kossa/van Gieson-stained sections.

For histological analysis, tibial bones were fixed in Histofix (Roth) for 12h and decalcified in EDTA (Sigma-Aldrich). Serial paraffin sections (2 μm) were stained for H&E and tartrate resistant acid phosphatase (TRAP) using a Leukocyte Acid Phosphatase Kit (Sigma) according as to the manufacturer’s instructions. Undecalcified bones were embedded in methacrylate and 5µm section were cut. Toluidine blue staining was performed for quantification of osteoblasts. Osteoclast and osteoblast numbers were quantified using a microscope (Nikon) equipped with a digital camera and an image analysis system for performing histomorphometry (Osteomeasure; OsteoMetrics).

Micro-CT images of the tibias were acquired as described previously [13] (link). Briefly, the tibias were harvested, fixed in 10% neutral buffered formalin for 24 h and stored in 70% ethanol. All quantifications were performed with digital image analysis (OsteoMeasure; Osteometrics, Atlanta, GA). The following parameters were measured: fraction of bone volume of the total sample volume (bone volume/tissue volume (BV/TV)), trabecular number (Tb.N), trabecular thickness (Tb.Th), and trabecular separation (Tb.Sp).
Histomorphometric analysis was performed on methacrylate-embedded undecalcified plastic sections of bone and were stained using von Kossa’s stain and TRAP stain. To measure bone formation rate (BFR), all mice were injected with calcein green solution (30 mg/kg, Sigma, MO) at 10 days and 2 days prior to harvesting. The tibias were prepared as described above and the measurements were performed using an image analysis system (OsteoMeasure) and the bone formation rate was calculated and the data are shown as µm³/µm²/year.

For immunofluorescence staining, sections were incubated with anti-HIF-1α antibody (1:50, H1a67, Novus) and anti-B220 antibody (1:100, RA3-6B2, Abcam) followed by incubation with AF488- and AF647-conjugated secondary antibodies (1:200, VECTOR). Fluorescence images were captured by a Zeiss confocal microscope. For histological analysis, serial paraffin sections were stained using an H&E staining kit (Carl Roth), Leukocyte Acid Phosphatase Kit (Sigma) or Toluidine Blue. For histomorphometry evaluation, a Nikon microscope equipped with a histomorphometry analysis system (OsteoMeasure; Osteometrics) was used. All samples were blinded for quantitative analysis.

Bone histomorphometry was performed using a microscope (Nikon) equipped with a digital camera and an image analysis system (OsteoMeasure; OsteoMetrics). The following parameters were measured: total size of the proliferation and specific of the hypertrophic chondrocyte part. Sections were scored by one independent observer (G. Schett) blinded for the specific mouse strain. No samples were excluded from analyses.