Dfc425b

The samples were fixed for 72 h in 10% formalin solution, dehydrated in ascending graded alcohols and embedded in LR White resin (Sigma-Aldrich) (Pizzicannella et al., 2011 (link)). Following polymerization, undecalcified oriented cut sections of 50 μm were obtained and ground down to about 30 μm by using the TT System (TMA2, Grottammare, Italy). The sections were analyzed with the CLSM LSM510 META (Zeiss) and, after staining with a solution of acid fuchsine and methylene blue, they were observed at light microscopy. The investigation was carried out by means of a bright-field light microscope (Leica Microsystem, Milan, Italy) connected to a high-resolution digital camera DFC425B Leica (Leica Microsystem).

In order to perform histological analysis, samples were fixed for 72 h in 10 % formalin solution, dehydrated in ascending graded alcohols and embedded in LR White resin (Sigma-Aldrich) [44 (link)]. After polymerization, undecalcified oriented cut sections of 50 μm were obtained and after ground down to about 30 μm using the TT System (TMA2, Grottammare, Italy). Sections were washed three times with distilled water and placed in silver nitrate solution (1%) under intense light for 3 h. Then, silver nitrate solution was removed and the scaffolds were washed again three times with distilled water. By adding sodium thiosulfate solution (5%) for 5 min, unreacted silver was removed from the scaffolds. Finally, the samples were washed with distilled water and observed by invert microscopy. The investigation was carried out by means of a bright-field light microscope (Leica Microsystem, Milan, Italy) connected to a high-resolution digital camera DFC425B Leica (Leica Microsystem).
In order to evaluate vascularization, the sections were observed under a light microscope after a double-staining procedure with methylene blue and fuchsin acid solutions.