Nonylphenyl polyethyleneglycol acetate

Static and dynamic histomorphometry was performed on decalcified and undecalcified femoral sections of mice.
For static analysis, femurs or tibias were fixed in 4% paraformaldehyde (Merck) for 3 days, washed for 2 hours with water, and decalcified in the presence of 15% EDTA for 10 days with mild shaking at 37°C. The bones were embedded in paraffin. Osteoclast number/bone perimeter and osteoclast surface/bone surface were quantified on decalcified sections of either tibia or femur stained for tartrate‐resistant acid phosphatase (TRAP).
Undecalcified femurs were first dehydrated and infiltrated with destabilized methylmetacrylate (Merck), benzoylperoxide (Merck), and nonylphenyl‐polyethyleneglycol acetate (Sigma‐Aldrich, St. Louis, MO, USA) for 14 days. Embedding in methylmetracrylate was done overnight at 4°C, and the femurs were used for dynamic analysis. Osteoblast number/bone perimeter, osteoblast surface/bone surface, and osteocyte number were quantified on toluidine blue‐stained undecalcified femoral sections. Two sections were analyzed for each mouse. Bone formation rate (BFR)/bone surface was measured on undecalcified bone sections by means of fluorochrome labeling. Osteomeasure software was used for the analysis (Osteometrics, Decatur, GA, USA)

To illustrate the micro-architecture of the callus, undecalcified bone histology was performed. The bones were fixed in 10% neutral buffered formalin for 14 days at room temperature, dehydrated with ascending concentration of ethanol and embedded in methylmetacrylate for 5 days at 4°C. Afterwards tissue was soaked in methylmethacrylat monomer, nonylphenyl-polyethyleneglycol acetate and azoisobutyronitrile (all Sigma-Aldrich, St. Louis, USA). The blocks were released from the glass vials and undecalcified sections of 40 μm were sawed and grinded using an EXAKT diamond saw system (Exakt, Norderstedt, Germany).

Tissue samples containing the screws were first cut into blocks of ∼30 mm thickness using an EXAKT precision saw (EXAKT Apparatebau, Norderstedt, Germany), followed by tissue fixation in 4% neutral-buffered formaldehyde. After fixation, the specimens were dehydrated in ascending grades of ethanol. Samples were then embedded into resin (methylmethacrylate, nonylphenyl-polyethyleneglycol acetate, benzoyl peroxide, Sigma-Aldrich, Merck KGaA, Darmstadt, Germany). Undecalcified thin-ground sections along the longitudinal axis of the screws in the frontal plane of the tibia shaft were produced according to the method of Donath [27 ], using EXAKT cutting and grinding equipment (EXAKT Apparatebau, Norderstedt, Germany). For Giemsa staining, polished thin ground sections were etched for 2 min in 0.1% formic acid, rinsed in distilled water, submersed in 20% methanol for 15 min and rinsed again in distilled water. Staining took place for 30 min in a 20% Giemsa solution (Merck KGaA, Darmstadt, Germany) in distilled water at 60°C. Two final rinsing steps were performed, consisting of rinsing with acidic solution (three drops acidic acid/100 ml distilled water) followed by distilled water. Samples were digitized with an Olympus BX53 microscope automatic system and the software OLYMPUS cellSens Dimension 3.1 (Olympus, Tokyo, Japan).