Axiocam 5 colour camera

Femora were fixed using formaldehyde 4% and embedded in paraffin blocks. After sectioning, samples were deparaffinized, hydrated, and stained in Alcian blue and Picrosirius red solutions [27 (link)]. Samples (n = 4) were observed using a Zeiss Axiolab 5 microscope and images were captured using an Axiocam 5 Colour Camera (Zeiss).

The angiogenic activity of L-PRF was evaluated using the chorioallantoic membrane (CAM) model [24 (link)]. The assay was performed according to Schmitd et al., with some modifications [25 (link)]. In short, fertilized chicken eggs (Gallus domesticus) were incubated in an Octagon 40 ECO rotating egg incubator (Brinsea, UK) at 37.5 °C, 50% humidity and one rotation per hour, for 8 days. The incubation was interrupted, and the air sac was delimited. The eggshell from the marked region was carefully removed, as well as the outer membrane of the egg. A section of the L-PRF membrane (5 × 10 mm) was placed on the exposed CAM and the egg was covered with a sterile transparent film. The eggs were then incubated in the same conditions for 2 days, without rotation. Finally, the CAM was removed with the L-PRF membrane and fixed with 4% formaldehyde for 4 h. The CAM was imaged with a Zeiss Stemis 305 stereo microscope. Images were captured using an Axiocam 5 Colour Camera (Zeiss, Jena, Germany). For the average vascular diameter measurement, the Vessel Analysis plugin of the Fiji software (v.1.53c) was applied [26 (link)]. Experiments were performed in triplicate and representative macrographs were acquired for all the conditions.