*2.1. Tissue Samples and Histology*

The National Research Ethics Service (11/NW/0875) gave ethical approval and informed written consent was obtained from patients undergoing autologous cell therapy for cartilage defects in their knee (*n* = 10, aged 29–51 years). This procedure entails harvesting ~200 mg macroscopically healthy cartilage, usually from the trochlea, from which chondrocytes are isolated and culture expanded in monolayer, prior to re-implantation in the defect site, usually on the patella or lateral/medial femoral condyles (LFC/MFC) [3]. At approximately 12 months post-implantation, full-depth cartilage biopsies with subchondral bone (1.8 mm diameter) were obtained using a juvenile bone-marrow biopsy needle from both the harvest site (naturally repaired) and the defect site where the cells had been implanted (cell-treated repair). The location of these was ensured via the use of knee maps [24], where the location of each procedure is recorded at the time of original surgery. Macroscopically healthy cartilage was also obtained from the knees of five cadavers (aged 21–63 years) and four donors undergoing total knee arthroplasty for OA (aged 51–81 years). A description of the donor demographics and tissue samples used in the following experiments can be found in Table 1. Healthy cadaveric cartilage from donors 11–14 was obtained within 24 h

of death from the UK Human Tissue Bank with approval by the Trent Research Ethics Committee (UK). Full-depth core biopsies of other samples (from TKR and natural and cell therapy repair patients) of cartilage and underlying bone were snap frozen within 2–4 h of harvesting in liquid nitrogen-cooled hexane and stored at −196 ◦C until cryosectioning. Cores were embedded into tissue-freezing medium (Leica) and cryosectioned at 7 µm thickness onto poly-L-lysine-coated slides. Cryosections were then stained with either haematoxylin and eosin (H&E) or toluidine blue for the assessment of general morphology and proteoglycan content of the cartilage, respectively. Collagen fibre organisation and orientation were assessed under polarised light. The quality of the repaired cartilage was assessed and scored semi-quantitatively using both the International Cartilage Repair Society II Histology Score (ICRS II) [25] and the Oswestry Score [26], where a higher score in each system represents better-quality cartilage.


**Table 1.** Donor demographics and samples.


**Table 1.** *Cont.*

FC = flow cytometry, IHC = immunohistochemistry, TKR = total knee replacement, ACI = autologous chondrocyte implantation, LFC = lateral femoral condyle, and MFC = medial femoral condyle.
