# Role of Autologous Platelet Gel (APG) in Bone Healing: A Rabbit Study
## 2 Materials and Methods
### Specim Processing  
The specimens were first washed in saline solution and after 10 min were put into 4% paraformaldehyde and 0.1% glutaraldehyde in a 0.15 M cacodylate buffer al 4 \({}^{\circ}\)C and pH 7.4, for fixation of the tissues and for processing to achieve specimen histology. SCAN 1 Automated System (Pescara, Italy) was used to achieve the sections containing the defects. The specimens were dehydrated in ascending concentrations of ethyl alcohol rinses, from 60% to 100%, and embedded in a glycolmethacrylate resin (Technovit 7200 VLC, Kulzer, Wehrheim, Germany). This resin was polymerized with UV light and the block was sectioned, along the longitudinal axis, with a diamond disc at about 140 um then ground down to about 40 um with a specially designed grinding machine to obtain 3 slides for each specimen. Tolulidine blue and acid fuchsin were used to stain the specimens as previously described [15]. The slides were then observed through normal transmitted light by a Nikon microscope ECLIPSE (Nikon, Tokyo, Japan). The microscope was connected to a high-resolution video camera with a high definition monitor and a computer workstation (Notebook Toshiba Satellite pro r50-c15w). A history software package for image capturing was used for morphometric analysis by digital image-analysis (NIS-Elements AR 3.0 software, Nikon, Minato, Japan). The aspects of the newly formed and mature bone could be classified due to the histological color of the tissues (light red = old matrix, dark red = new matrix) and their quantity was expressed in percentage (Mean \(\pm\) SD). Tolulidine blue and acid fuchsin staining revealed cell types and patterns of hard and soft tissue present in the defects. Newly formed bone was distinguishable from the original bone due to a higher intensity of staining in the new bone. For the histomorphometric analysis, a region was selected for a representative measurement. The selected region of interest (ROI-1) 6000 \(\times\) 2500 \(\mu\) included the whole defect area containing the newly formed bone. For the evaluation of medullary space, a region of interest (ROI-2) 3000 \(\times\) 3000 \(\mu\) containing the central portion of tibia was selected. Newly formed bone was distinguishable from the original bone due to a higher intensity of staining in the new bone. After acquiring the image to be analyzed, the first step in software analysis was to identify the region of interest manually ROI, using the overlay drawing tools. Next, the bone tissue was identified by an interactive threshold in the hue-saturation-brightness (HSB) colour space. If necessary, an option can be used that allows the use of a hysteresis threshold  
Figure 1: (**A**) Two bone defects in the rabbit tibia. (**B**) The test bone defect filled with autologous platelet gel (APG).  
for the saturation band. The small sectioning artefacts, such as cracks, can be removed using simple binary close ('close cracks') and hole-fill operations. Small fragments in the image are removed using an erode-reconstruct algorithm. The resulting image allowed for the quantitative measurement of the mineralized bone and quantity of marrow space and red intensity was digitally image-analyzed. The data obtained from the analyses were transformed into absolute values of pixels and then to percentages.  
The following measurements were taken: Lamellar bone surface: Expressed as percentages of the region of interest; woven bone surface: Expressed as percentages of the region of interest; and marrow space surface: Expressed as percentages of the region of interest.