Histomorphometric Analysis of Fracture Callus

Mice were killed at 3, 5, 7, 10, 14, 18, 21, 25, 30, or 35 days after fracture. A normal mid-diaphysis femoral bone segment was used as a nonfractured day 0 control. Femurs were disarticulated from the hip and trimmed to remove excess muscle and skin. Specimens were stored in 10% neutral buffered formalin for 2 days. The tissues were infiltrated and embedded in paraffin. Alcian blue and orange G along with TRACP staining was done as previously described.(19 (link),20 (link),28 (link)) Histomorphometric analysis (n = 4 animals per group) was done using a standardized eyepiece grid to measure tissue areas within the fracture callus. Samples were cut at four levels spanning ∼120 μm through the callus, with 30 μm between each level. Each cross-hatch was categorized as not callus (not counted), callus (quantified), and a specific tissue type. A total area of the external callus and areas of individual tissue types such as new bone (mineralized tissue), total cartilage, immature proliferative cartilage, hypertrophic cartilage, and mesenchyme were quantified. Bone was defined as areas of new woven bone. Cartilage was defined as tissues staining blue for proteoglycan. Hypertrophic cartilage was clearly defined by cellular morphology, and other nonhypertrophic areas of cartilage were considered immature cartilage. Finally, mesenchyme was defined as areas containing spindle-shaped fibroblasts without Alcian blue staining. Cortical bone was excluded from the histomorphometric analysis. The target tissue area was divided over the area of the external callus.
In the rescue experiment, aged mice were treated with either vehicle or a nonprostanoid EP4 selective agonist, CP432 (Pfizer, Groton, CT, USA). CP734432 (CP73) was freshly prepared in normal saline containing 3% ethanol. CP73 (100 μl) was injected at the fracture site twice daily for a total daily dose of 20 mg/kg/d.