*2.7. Histological Analysis*

Femurs were collected and fixed in 10% bu ffered formalin for 24 h at 4 ◦C and were decalcified with 10% neutralized EDTA (Sigma) for 4 weeks; then dehydrated with an ascending series of ethanol concentrations, cleared in xylene and embedded in Paraplast (Sigma). Histological sections (6 μm) were stained with hematoxylin-eosin (H&E) for general morphologic analysis or picrosirius for collagen fibers (type I and type III) quantification and stereological analysis [50]. The color displayed under polarizing microscopy was a result of fiber thickness, as well as the arrangemen<sup>t</sup> and packing of the collagen molecules. Normal tightly packed thick collagen fibers had polarization colors in the red spectrum while thin or unpacked fibers had green birefringence [51]. Sections were observed under normal and polarized light, and digitalized images were analyzed using Leica Q-win software (Version 3.0) to calculate mean collagen fiber area.

Non-injured bone was used to show di fferences with our injured groups in radiographic evaluation and histological analysis (H&E and picrosirius).

### *2.8. Scanning Electron Microscopy (SEM)*

SEM analyses were performed using a Quanta 200 electron microscope (FEI Company, Hillsboro, OR, USA). Bone samples were fixed in 2.5% glutaraldehyde in 0.1-M PBS pH 7.3 for 4 h. Samples were then removed and washed three times for 5 min in distilled water. Subsequently, samples were immersed for approximately 40 min in 0.5% osmium tetroxide and washed three times in distilled water; dehydrated in increasing concentrations of ethanol (7.5% to 100%); dried in a critical point apparatus with liquid carbon dioxide, mounted on appropriate chucks, metallized and gold-coated [37].
