Von kossa

Hindlimbs were fixed in 4% formaldehyde (fromPFA)) for 72hrs at 4°C before being decalcified 0.5M ethylenediaminetetraacetic acid (EDTA) pH7.4 at 4°C. Samples were embedded in paraffin wax blocks and 5μm sagittal sections cut. For cryotome sectioning, samples were equilibrated in a 30% sucrose/phosphate buffered saline (PBS) solution at 4°C and then embedded in OCT compound (Fisher Scientific, Loughborough, UK) before 10μm sagittal sections were cut. For human material, 8x3mm blocks of AC were cut from femoral tibial condyles and fixed in neutral buffered formalin and then paraffin wax embedded. Histological staining with Von Kossa (Abcam, Cambridge, UK), toluidine blue (Sigma) and haematoxylin and eosin (Sigma) were carried out according to standard procedures. All histological scoring was carried out on the lateral tibial condyle with AC damage determined by a binary scoring system, of ‘normal’ or ‘damaged’. At least three slides separated by 25μm were analysed for each limb. For cell and immunohistochemical scoring, cell-types or positive staining cells were expressed as a percentage of the total chondrocyte count. The number of empty lacunae were expressed per mm of AC analysed.

Post micro-CT analysis, three samples of each scaffold type were rinsed in PBS and then embedded in Tissue-Tek^® O.C.T. compound (Sakura Finetek, The Netherlands) and stored at −80°C. Slices measuring 14 µm in thickness were sectioned using a CM1900 microtome (Leica) and mounted to glass slides. Slides were then stained with Hematoxylin (Leica) and Eosin (Thermo Fisher Scientific), Alizarin Red S (Sigma-Aldrich) or Von Kossa (Abcam, Cambridge, UK). Immunohistochemistry was performed using a polyclonal antibody to osteopontin (OPN, ab8448, Abcam) and methyl green (Sigma-Aldrich) with a goat pAB to Rb IgG (HRP) (ab6721, Abcam) secondary antibody in normal goat serum (Jackson ImmunoResearch, West Grove, PA, USA). methyl green (Sigma- Aldrich) was used as a counterstain. All histologically stained slides were analyzed qualitatively and imaged using a NanoZoomer Digital Pathology System (Hamamatsu, Japan).

To determine matrix mineralisation following osteoblast-like cell differentiation, cells were stained with von Kossa (Abcam, Cambridgeshire, UK) according to the manufacturer’s instructions. Calcium deposition was detected by incubating the cells with 2% Alizarin red S pH 4.2 for 5 min. Hydroxyapatite deposition was detected using the OsteoImage bone mineralisation assay (Lonza, Slough, UK).