Histology and Von Kossa staining

To assess deposition of CaP throughout the depth of the FA scaffolds, histology followed by Von Kossa staining was carried out. Samples were processed, embedded in paraffin wax and sectioned into 4 µm thick sections. The sections were adhered to glass slides and placed in a hotbox at 60°C for 25 min. Once the sections were dry, Von Kossa staining was conducted. The principle of the Von Kossa staining is a precipitation reaction in which silver ions react with phosphate under acidic conditions. Then, photochemical degradation of silver phosphate to silver occurs under light illumination. Slides were covered with 1.5% silver nitrate solution and exposed to bright light for 1 h (under a lamp), after which they were washed with dH₂O. Then, slides were covered with 2.5% sodium thiosulphate for 5 min and dipped in running water before immersion in Eosin counter stain for 5 min. Slides were dipped in 70% IMS, then 90% IMS and immersed in 100% IMS for 1 min. Finally, they were immersed in Xylene for 2 min, dipped twice in Xylene and then a coverslip was placed using DPX mounting media, for observation under light microscopy. CaP deposits were stained black/dark brown while FA fibres were stained pink/red.